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. 2012 Sep 18;153(11):5406–5414. doi: 10.1210/en.2012-1357

KNDy (Kisspeptin/Neurokinin B/Dynorphin) Neurons Are Activated during Both Pulsatile and Surge Secretion of LH in the Ewe

Christina M Merkley 1,*, Katrina L Porter 1,*, Lique M Coolen 1, Stanley M Hileman 1, Heather J Billings 1, Sara Drews 1, Robert L Goodman 1,**, Michael N Lehman 1,**,✉
PMCID: PMC3473209  PMID: 22989631

Abstract

KNDy (kisspeptin/neurokinin B/dynorphin) neurons of the arcuate nucleus (ARC) appear to mediate the negative feedback actions of estradiol and are thought to be key regulators of pulsatile LH secretion. In the ewe, KNDy neurons may also be involved with the positive feedback actions of estradiol (E2) to induce the LH surge, but the role of kisspeptin neurons in the preoptic area (POA) remains unclear. The goal of this study was to identify which population(s) of kisspeptin neurons is (are) activated during the LH surge and in response to the removal of E2-negative feedback, using Fos as an index of neuronal activation. Dual-label immunocytochemistry for kisspeptin and Fos was performed on sections containing the ARC and POA from ewes during the luteal phase of the estrous cycle, or before or after the onset of the LH surge (experiment 1), and from ovary-intact, short-term (24 h) and long-term (>30 d) ovariectomized (OVX) ewes in anestrus (experiment 2). The percentage of kisspeptin neurons expressing Fos in both the ARC and POA was significantly higher during the LH surge. In contrast, the percentage of kisspeptin/Fos colocalization was significantly increased in the ARC, but not POA, after both short- and long-term E2 withdrawal. Thus, POA kisspeptin neurons in the sheep are activated during, and appear to contribute to, E2-positive feedback, whereas ARC kisspeptin (KNDy) neurons are activated during both surge and pulsatile modes of secretion and likely play a role in mediating both positive and negative feedback actions of E2 on GnRH secretion in the ewe.

Although the importance of GnRH in reproduction is well known, identification of the pathways through which estradiol (E2) regulates GnRH release remains an area of continuing investigation. Ovarian steroids exert negative feedback on GnRH/LH secretion throughout the luteal and early follicular phase of the estrous cycle (1). During the breeding season of ewes, E2 serves to inhibit LH pulse amplitude whereas progesterone inhibits pulse frequency (1, 2); however, during anestrus (nonbreeding season), E2 inhibits LH pulse frequency (3). In contrast to these inhibitory effects, the preovulatory GnRH/LH surge that occurs at the end of the follicular phase is induced by high levels of E2 secreted from preovulatory follicles (4). Estrogen-receptor α (ERα) is the key receptor in mediating regulation of GnRH by E2 (5). Because few, if any, GnRH neurons express ERα (6), the feedback actions of E2 must occur via other neurons, and recent work points to kisspeptin neurons as playing a key role in this function. Importantly, most kisspeptin neurons express ERα (7) and can directly interact with GnRH neurons, 90% of which express the kisspeptin receptor (8–10).

Kisspeptin, a member of the RF-amide family of neuropeptides, binds to its receptor, GPR54 (11, 12), and stimulates LH release in humans (13) as well as in mice (14), rats (15, 16), sheep (17), and primates (18). A mutation in GPR54 causes hypogonadotropic hypogonadism in humans (19, 20), indicating the importance of the kisspeptin-GPR54 interaction in controlling sexual maturation and reproduction. Kisspeptin has now been identified as a key regulator of GnRH and LH secretion and has been proposed to mediate both negative and positive feedback actions of E2 (21, 22).

Two anatomically distinct kisspeptin populations exist in rodents. Those in the anteroventral periventricular nucleus (AVPV) have been proposed to mediate the E2-induced GnRH/LH surge (23, 24), whereas kisspeptin neurons in the arcuate nucleus (ARC) are thought to be responsible for E2-induced inhibition of episodic GnRH/LH secretion (21, 24–26). Sheep do not have a distinct AVPV, but kisspeptin neurons are evident in both the preoptic area (POA) and ARC, with some anatomical differences between the two populations. Almost all kisspeptin neurons in the ARC coexpress dynorphin and neurokinin B and, hence, are referred to as KNDy neurons (27). More than 90% of these neurons receive input from other KNDy neurons, thus forming an interconnected, reciprocal network with one another (28–30). POA kisspeptin neurons in sheep do not exhibit colocalization with dynorphin or neurokinin B, and do not exhibit the same degree of reciprocal connections with each other as seen among KNDy neurons. There also may be functional differences between these two kisspeptin-containing neural populations. The effects of E2 removal (22) and replacement (10, 22) on Kiss1 mRNA expression implicate ARC kisspeptin neurons as important mediators of E2-negative feedback. However, the role of both POA and ARC kisspeptin neurons in the preovulatory LH surge remains unclear. During late follicular phase, elevated Kiss1 mRNA expression has been observed in both the POA and some regions of the ARC (10). However, studies using Fos to analyze neuronal activation of kisspeptin neurons have been inconclusive (10, 31). The purpose of this study was to determine which population(s) of kisspeptin neurons are activated during the LH surge and in response to removal of E2-negative feedback, using Fos as an index of neural activity. Because anatomical subsets of ARC kisspeptin neurons have been implicated in the surge (10), and to assess the possibility that different ARC kisspeptin neurons might be dedicated to surge and tonic GnRH secretion, we analyzed Fos expression in rostral, middle, and caudal portions of the ARC, as has been done in previous work (10).

Materials and Methods

Animals

Adult blackface ewes were maintained under ambient conditions in an open barn with free access to food and water. Three to five days before any experimental procedures, ewes were moved to an indoor facility with controlled photoperiod simulating natural outdoor day length. In this facility, they had free access to water and a mineral lick and were fed a pelleted maintenance diet daily. Estrous behavior was determined using vasectomized rams. Blood samples (3–5 ml) were taken by jugular venipuncture and placed into heparinized tubes, and plasma was collected and stored at −20 C. Ewes used for experiment 1 were killed during the breeding season (October through February), and ewes used in experiment 2 were killed during the nonbreeding season (May through July). All experimental procedures involving animals were approved by the West Virginia University Animal Care and Use Committee and followed the National Institutes of Health guidelines for animal research.

Animal protocol

Experiment 1: which kisspeptin neurons are activated during the preovulatory LH surge?

To collect tissue from ewes at specific times of the estrous cycle, their cycles were synchronized with im injections (10 mg) of prostaglandin F2α (PGF2α; Lutalyse; Upjohn, Kalamazoo, MI) to induce regression of the corpus luteum. Once cycles were synchronized, brain tissue was collected on d 9–d 10 of the luteal phase (n = 4) or PGF2α was again injected and two controlled internal drug releasing (CIDR) devices were inserted to maintain luteal phase progesterone concentrations (32). The CIDRs were removed 10 d later, and blood samples were collected every 4 h starting 12 h after CIDR removal until perfusion. Tissue was collected 4 h after onset of estrous behavior and, based on LH concentrations, animals were classified into presurge (n = 11) and surge (n = 5) groups. One ewe included in the surge group was estrus-synchronized with only PGF2α (i.e. CIDRs omitted) and was killed during a clear LH surge.

Experiment 2: which kisspeptin neurons respond to removal of E2-negative feedback?

This experiment was performed in anestrus, when E2 is the only ovarian steroid inhibiting GnRH pulse frequency (2, 3). Bilateral ovariectomies (OVX) were performed via midventral laparotomy using sterile procedures on 10 anesthetized (isofluorane) ewes as previously described (33). Tissue from the short-term OVX group (n = 5) was collected at 24 h post-OVX, and tissue from the long-term OVX group (n = 5) was collected at least 1 month after OVX. Tissue from ovary-intact anestrous ewes (n = 5) was also collected. Blood samples were collected every 12 min for 4 h just before tissue collection in all three groups to determine LH pulse patterns.

Tissue collection

Tissue collection and processing were performed as previously described (27). Ewes were euthanized via an iv overdose of sodium pentobarbital (∼2 g in 7 ml saline; Sigma, St. Louis, MO) after two iv injections of heparin (25,000 U), 10 min before and immediately before pentobarbitol. The head was removed and perfused through both carotid arteries with 6 liters of 4% paraformaldehyde containing 10 U/ml heparin and sodium nitrate. After perfusion, brains were removed, and POA/hypothalamic tissue was dissected out. The tissue was then infiltrated with 30% sucrose, and coronal sections (50 μm thick) were cut on a freezing microtome and stored at −20 C in cryoprotectant for later processing.

Assays

LH was measured in duplicate aliquots of 50–200 μl of plasma, using a previously validated RIA (33), and expressed in terms of NIH-LH-S12. The minimal detectable concentration of LH in these assays average 0.077 ng/tube; inter- and intraassay coefficients of variation were 3.8% and 1.7%, respectively. Circulating progesterone was measured in duplicate aliquots of 150 μl plasma using a commercially available solid-phase RIA kit (Coat-A-Count P4; Diagnostic Products Corp., Los Angeles, CA), which has been validated in sheep (34) to confirm stage of cycle.

Immunocytochemistry for kisspeptin and Fos

All immunohistochemistry was carried out on free-floating sections at room temperature, and sections were washed with 0.1 m PBS between incubations. For all experiments, tissue sections were first incubated in hydrogen peroxide (10 min in PBS; EMD Chemicals, Inc., Gibbstown, NJ) to occupy endogenous peroxidase sites, followed by incubation in a solution containing 20% normal goat serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS containing 0.4% Triton X-100 (Fisher Scientific, Pittsburgh, PA) for 1 h to minimize nonspecific binding.

A dual-label immunoperoxidase procedure was then performed on a series of every fifth POA and ARC section in each animal. Fos-ir nuclei were detected using a rabbit polyclonal antibody against c-Fos [1:1,000(Exp. 1) or 1:2500 (Exp. 2); sc-52, Santa Cruz Biotechnology, Inc., Santa Cruz, CA] for 17 h, followed by incubation with biotinylated goat antirabbit IgG (1:500; Jackson ImmunoResearch Laboratories, Inc.) for 1 h and avidin-biotin-horseradish peroxidase complex (ABC-elite; 1:500; Vector Laboratories, Burlingame, CA) for 1 h. Labeling was visualized using nickel-enhanced diaminobenzidine (Sigma) as the chromogen, producing a black/purple reaction product that is limited to the nucleus. For detection of kisspeptin, sections were next incubated for 17 h in polyclonal rabbit antikisspeptin-10 serum (1:100,000 (Exp. 1) or 1:50,000 (Exp. 2); no. 564, gift from A. Caraty, Université Tours, Nouzilly, France], an antibody that has been previously validated for use in sheep tissue (27, 35). Next, sections were incubated with biotinylated goat antirabbit IgG (1:500; Jackson ImmunoResearch Laboratories, Inc.) and ABC-elite (1:500; Vector Laboratories) for 1 h each. Kisspeptin was visualized using diaminobenzidine as the chromogen, which produced a brown reaction product within the cytoplasm. Sections were then mounted onto Superfrost slides, air-dried, dehydrated, and coverslipped using Depex (DPX; Electron Microscopy Sciences, Hatfield, PA).

For each ewe, the numbers of single kisspeptin and dual-labeled kisspeptin/Fos cells were counted under bright-field illumination in the POA (three sections per ewe) and ARC (three sections per rostral, middle, and caudal subdivisions in each ewe) by an independent observer. POA sections selected for analysis were those at the rostral-caudal level of, and just posterior, to the organum vasculosum of the lamina terminalis. For selection of ARC sections, the rostral ARC was defined as that level of the nucleus anterior to the appearance of the tubero-infundibular sulcus; the middle ARC was at the level of the tubero-infundibular sulcus; and the caudal ARC was at the level of the mammillary recess of the third ventricle. To assess the possibility of functionally distinct subsets of ARC kisspeptin neurons, we analyzed all three rostral-caudal levels of the ARC to account for the possibility that there may be regional differences within the ARC in the functional activation of kisspeptin cells during the surge. To assist with analysis, bright-field images were captured using a cooled charge-coupled device camera (Microfire; Optronics, Goleta, CA) attached to a Leica microscope (DM5000B; Leica Microsystems, Weztler, Germany) and Neurolucida software (MicroBrightfield, Inc., Williston, VT) with fixed camera settings for all animals; a transparent grid was then placed over the printed images to aid with counting. A cell was considered dual labeled when brown kisspeptin-positive cytoplasm was seen to surround a black Fos-positive nucleus in the same plane of focus, and the percentage of dual labeling was calculated as the total number of dual-labeled cells divided by the total number of kisspeptin-positive cells.

Statistical analysis

The mean number of single-labeled kisspeptin and dual-labeled kisspeptin/Fos cells in each region (POA and rostral, middle, and caudal subdivisions of the ARC) were counted for each animal, and the percentage of kisspeptin cells containing Fos was calculated. The mean and sem were calculated for each group, and one-way ANOVAs were conducted within each region between luteal, presurge, and surge groups (Exp. 1), and intact, 1 d OVX, and chronic OVX groups (Exp. 2). Post hoc analyses of all pair-wise comparisons were computed using Tukey tests to account for the problem of multiple comparisons. In experiment 2, LH pulses were identified using standard criteria (1), and statistically significant differences in mean LH concentrations were analyzed by one-way ANOVA and in LH pulse frequencies by Kruskal-Wallis one-way ANOVA on ranks.

Results

Experiment 1: which kisspeptin neurons are activated during the preovulatory LH surge?

As expected, mean LH concentrations at perfusion in surge animals increased by 7- and 5-fold from luteal and presurge groups, respectively (P < 0.05) (Fig. 1B).

Fig. 1.

Fig. 1.

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A, Representative LH profiles from ovary-intact luteal, presurge, and surge ewes taken before perfusion. Note that on the y-axis of the presurge and surge LH profiles, time = 0 represents 12 h after CIDR removal. B, Mean LH levels at perfusion were significantly higher in the surge group compared with both luteal and presurge groups. Letters (a and b) represent significant differences from other groups, P < 0.05.

Anatomically, kisspeptin perikarya were localized to the POA and ARC, with the greatest numbers of cell bodies seen within the middle ARC and the fewest number of cell bodies in the POA (Fig. 2A). In the middle ARC, presurge animals had significantly greater numbers of kisspeptin cells compared with luteal phase animals, but not surge animals (P < 0.05; Fig. 2A). There were no significant effects of stage of cycle on kisspeptin cell numbers in any other area. In addition, we noted that labeling of kisspeptin-ir fibers appeared more intense in the presurge animals compared with the surge animals (Fig. 2C).

Fig. 2.

Fig. 2.

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Kisspeptin cells in both the ARC and POA are activated during the LH surge. Kisspeptin cell number (A) and percent colocalization with Fos (B) in luteal, presurge, and surge groups in the POA, and rostral, middle, and caudal levels of the ARC. Letters (a, b, and c) represent significant differences between groups within the same area, P < 0.05. C, Sections immunostained for kisspeptin and Fos through the middle ARC (left column) and POA (right column) from luteal, presurge, and surge animals. Arrows show examples of dual-labeled cells. Scale bar, 10 μm.

Surge animals showed a significantly greater percentage of kisspeptin cells colocalizing Fos compared with both luteal and presurge animals throughout the rostral, middle, and caudal divisions of the ARC (P < 0.05; Fig. 2B) and in the POA (P < 0.05; Fig. 2B). The percentage of kisspeptin cells that colocalized Fos in each region of surge animals was consistent, ranging from 44% to 61% in the both ARC (rostral, middle, caudal) and POA, but because there were far fewer kisspeptin cells in the POA, the absolute number of Fos-expressing cells in this region was lower than in the ARC (Fig. 3). Examples of single- and dual-labeled kisspeptin neurons in the ARC and POA are shown in Fig. 2C, and their anatomical location is shown in Fig. 3. The percentage of colocalization of kisspeptin with Fos did not differ between luteal and presurge animals in any area (Fig. 2B).

Fig. 3.

Fig. 3.

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Camera lucida drawings of representative sections through the POA (top) and ARC (bottom) of sheep killed either during the LH surge (right) or 30 d after OVX (left). The location of single-labeled kisspeptin cells (open circles) and dual-labeled Fos/kisspeptin cells (black circles) are shown at low and high magnification in each of the drawings (boxed areas). ac, Anterior commissure; 3V, third ventricle; fx, fornix; oc, optic chiasm; ot, optic tract; mt, mammillothalamic tract.

Experiment 2: which kisspeptin neurons respond to removal of E2-negative feedback?

As expected, LH pulse frequency increased after OVX from 1.0 ± 0.3 pulse/4 h in intact ewes to 2.0 ± 0.3 pulses/4 h 1 d after OVX. Frequency further increased to 4.0 ± 0.4 pulses/4 h in chronically OVX animals (Fig. 4). Mean LH concentrations also increased 3- and 9-fold in 1-d OVX and long-term OVX ewes, respectively (Fig. 4B).

Fig. 4.

Fig. 4.

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A, Representative LH profiles from ovary-intact, 1 d after OVX, and more than 30 d post-OVX ewes. Black circles indicate pulse peaks. Note different scale on y-axis for data from ewe OVX for more than 30 d. B, Mean pulse frequency (top panel) and LH concentration (bottom panel) in each group of ewes. Letters (a, b, and c) represent significant differences from other groups, P < 0.05.

In the ovary-intact anestrous ewes, ARC kisspeptin cells were most numerous in the caudal ARC, with moderate numbers present in the middle ARC and POA (Fig 5A). After more than 1 month after OVX, kisspeptin cell numbers significantly increased in both the rostral and middle ARC.A similar pattern of increased cell numbers was also observed in the 1-d post-OVX ewes (P < 0.05; Fig. 5A). There were no significant effects of OVX on kisspeptin cell numbers in the POA.

Fig. 5.

Fig. 5.

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Ovariectomy increases activation of kisspeptin cells in the ARC, but not the POA. Kisspeptin cell number (A) and percent colocalization with Fos (B) in ovary-intact, 1 d post-OVX, and more than 30 d post-OVX ewes in the POA and rostral, middle, and caudal sections of the ARC. C, Letters (a, b, and c) represent significant differences from each other within the same area, P < 0.05. C, Sections immunostained for kisspeptin and Fos through the middle ARC in gonadal-intact and long-term (>30 d) OVX ewes. Arrowhead and arrows indicate examples of single- and dual-labeled cells, respectively. Scale bar, 10 μm.

E2 withdrawal significantly increased the percentage of ARC kisspeptin cells colocalizing Fos in the rostral, middle, and caudal ARC after 24 h post-OVX, and this colocalization was further increased in chronically OVX ewes (P < 0.05, Fig. 5B; and Fig. 3). No significant changes in the percentage of kisspeptin/Fos colocalization were observed in the POA at either time point, and this percentage of colocalization remained low (<10%).

Discussion

Using a well-defined surge model, we observed that kisspeptin neurons of both the ARC and POA are activated after the onset of the preovulatory surge, indicating a role for both populations in E2-positive feedback in the ewe. In contrast, we observed that ARC, but not POA, kisspeptin neurons are activated after the withdrawal of E2, suggesting that only ARC kisspeptin neurons are likely important mediators of E2-negative feedback.

Several lines of evidence support the involvement of both ARC and POA kisspeptin neurons in E2 positive feedback in the ewe. First, in the late follicular phase, Kiss1 mRNA has been shown to increase in the middle and caudal ARC, as well as in the POA (10, 36). Second, there was also an increase in kisspeptin protein in the ARC during this time (10).Third, E2 treatment of OVX ewes increased kisspeptin mRNA and protein expression in the POA (35). Leading up to the surge, we observed an increase in kisspeptin cell number and fiber intensity in the middle ARC, which may reflect an increased sequestration of the peptide in the cell body as a result of the up-regulated Kiss1 mRNA (10, 36).

Although the aforementioned data show that Kiss1 mRNA and kisspeptin protein are up-regulated before the surge, our data show that soon after the onset of the preovulatory surge, kisspeptin cells are transcriptionally activated in both the ARC and POA. Interestingly, Smith et al. (10) failed to show an increase in Fos expression within POA kisspeptin cells 1 h after a surge-inducing injection of E2 to OVX ewes. In their paper, they propose that the activation of these neurons may occur outside of the time window they chose to study (10), which could be consistent with our results. In contrast to the findings of Smith et al. (10), a recent study using ovary-intact ewes observed Fos expression in POA, but not ARC, kisspeptin neurons during the preovulatory LH surge (31). These authors suggested that the contrasting findings to previous work (10) might be due to different animal models (ovary-intact vs. OVX). Our data confirm the activation of POA kisspeptin at the time of the LH surge reported by Hoffman et al. (31), but we also observed increased Fos expression in the ARC population. One possible explanation for this discrepancy is that the chronic guide tubes traversing the ARC (used to monitor GnRH in the median eminence) in the animals used by Hoffman et al. (31) may have disrupted activation of ARC kisspeptin neurons. Another possible explanation is that different antibodies for detection of Fos were used in these two studies, suggesting the possibility that differences in observed Fos may be due to a differential antibody sensitivity (Smith et al. used the same antibody we did). Despite this discrepancy, most mRNA studies in the ewe are consistent with the idea that both ARC (KNDy) and POA kisspeptin neuronal populations participate in mediating E2 positive feedback in this species (10, 36).

In the second experiment, we observed a significant increase in both numbers of kisspeptin-positive cells and their activation (as measured by Fos) in the ARC of long-term OVX ewes. Kisspeptin cell numbers and activation were also elevated, coincident with increased LH release, as early as 24 h post-OVX, demonstrating the rapid response of ARC kisspeptin neurons to the removal of E2. No changes were observed in the POA kisspeptin neurons at either time point. Thus we suggest that ARC, not POA, kisspeptin neurons mediate E2-negative feedback and therefore are likely key regulators of episodic LH secretion. These data confirm previous studies that found that chronic removal of E2 by OVX increased (22), and chronic E2 treatment decreased, both Kiss1 mRNA expression (10, 22) and number of kisspeptin-ir cells in the ovine ARC (10) during the breeding season. Our data extend these observations by demonstrating an early increase in both kisspeptin cell number and Fos colocalization after OVX. Similar to the work in sheep, studies in both humans and rodents also point to ARC kisspeptin neurons as mediators of E2-negative feedback. Postmenopausal women show both hypertrophy of kisspeptin neurons and increased kisspeptin gene expression in the infundibular nucleus (37), suggesting that kisspeptin in this area is normally held in check by steroid-negative feedback. In rodents, OVX increases, and E2 treatment decreases, Kiss-1 expression in the ARC (26), but there is no evidence that OVX increases Fos expression in these neurons (38).

In comparing kisspeptin populations involved in the positive and negative feedback actions of E2 among species in which this question has been addressed, two consistent patterns emerge: 1) the more rostral population (POA in sheep, AVPV and associated areas in rodents) is dedicated to the initiation of the LH surge, and 2) the ARC kisspeptin population participates in the negative feedback control of pulsatile LH secretion. In sheep, between 40% and 60% of POA kisspeptin neurons are activated during the surge (this study and Ref. 31). This roughly corresponds with the approximately 50% of POA kisspeptin-ir neurons that express ERα (7), but whether this population overlaps with the activated kisspeptin cells observed here remains to be determined. These neurons may be stimulated directly by E2, but this seems unlikely because in the ewe the mediobasal hypothalamus, not the POA is the site at which E2 acts to induce an LH surge (39). Alternatively, these cells may be indirectly regulated, and subsequently activated, by steroid-sensitive afferents. Although fewer kisspeptin cells in the POA than in the ARC expressed Fos, one cannot infer that the POA input is quantitatively less important until information on the number of GnRH neurons receiving efferents from these two populations is determined. In rodents, E2 increases both kisspeptin expression (26) and Fos colocalization with kisspeptin in the AVPV (24, 38, 40). Interestingly, the percentage (ranging from 30–60%) of rostral kisspeptin neurons activated at the time of the surge in rodents (24, 38, 40) is similar to that in sheep. Almost all of these neurons in rodents contain ERα (26), and evidence suggests that E2 positive feedback in rodents occurs directly on these kisspeptin neurons (41). Studies that examined the more rostral kisspeptin population observed relatively low levels of kisspeptin/Fos colocalization in the absence of an LH surge in both sheep (10, 31) and rodents (24, 38, 40), again consistent with this population being dedicated to the LH surge.

It is also generally accepted that the ARC kisspeptin (KNDy) neurons in rodents and sheep mediate the negative feedback actions of E2. It has also been proposed that they drive episodic GnRH secretion (42–44), but there may be some species differences in their activation. As already noted, OVX increased Fos expression in these neurons in sheep (this study), but apparently not in rodents (38). Moreover, in both current experiments, we observed that approximately 20% of KNDy neurons contain Fos during the follicular phase or during anestrus, even though LH secretion is at a basal level. This finding is apparently inconsistent with that of Hoffman et al. (31), who reported only about 5% of ARC kisspeptin neurons are activated in early follicular phase ewes (31). As discussed above, this may reflect differences in the antibodies used to detect Fos immunoreactivity or that Hoffman et al. (31) used tissue from ewes with chronic guide tubes traversing the ARC. Smith et al. (10) observed approximately 15% ARC kisspeptin/Fos colocalization during the luteal phase of ewes, which increased to about 35% during the late follicular phase. The relatively high level of Fos expression in ovine KNDy neurons, compared with POA kisspeptin neurons, during basal secretion of LH is consistent with the hypothesis that KNDy neurons are important in tonic secretion of GnRH (42–44). In contrast, colocalization of kisspeptin and Fos in the ARC of rats less than 5% on diestrus 2 of the cycle (24).

The one major difference between KNDy neurons in sheep and rodents is that they appear to participate in the LH surge in the former, but not the latter. Two of three studies observed high levels of Fos expression in KNDy neurons associated with the surge in sheep, whereas, with one exception (23), this has not been observed in rodents (24). As noted above, this difference is consistent with evidence that the positive feedback action of E2 occurs in the mediobasal hypothalamus in the sheep, and in the region of the AVPV in rodents (41). Interestingly, although almost all ovine KNDy neurons express ERα in the ewe (7), only 45–60% are activated during the surge. What remains unclear from these data are if this activation is due to heterogeneity in the ARC population, so that different subsets of neurons govern positive and negative feedback, or alternatively, if the same neurons govern positive and negative feedback, but are not activated at the same time. Moenter et al. (45), showed that approximately 40% of GnRH neurons were activated during the surge and postulated that different neurons may be activated over time throughout the GnRH surge to maintain high levels of GnRH release. The high level of Fos expression in KNDy neurons of OVX ewes (70–90%, Fig. 4) suggests that the same neurons are involved in both E2-positive and -negative feedback. Reciprocal connections between KNDy neurons, which are postulated to play an important role in episodic GnRH secretion (42–44), may be responsible for the high percentage of activated KNDy neurons after OVX. If this is the case, the lower percentage of KNDy neurons containing Fos during the LH surge, may suggest a less important role for this reciprocal innervation. This proposal is consistent with the changes in multiunit electrical activity associated with LH pulses at the time of the LH surge in monkeys, rats, and goats (46–48). Fos induction in most neurons is thought to be a consequence of glutamate release and its postsynaptic action, and it is interesting in this regard that a majority of KNDy neurons in the sheep colocalize glutamatergic markers (49), including expression within terminals that form reciprocal KNDy-KNDy contacts (50). Thus, release of glutamate from KNDy presynaptic terminals may be partly responsible for the Fos induction seen during increased pulsatile activity.

In summary, these data demonstrate that both the ARC (KNDy) and POA kisspeptin neurons are activated during, and therefore likely play an important role in, the GnRH/LH surge in ewes. In contrast, only ARC kisspeptin neurons had increased kisspeptin expression and activation after E2 removal, indicating the key role of ARC kisspeptin in the negative feedback regulation of episodic GnRH secretion by E2. Whether the same or different subsets of ARC kisspeptin neurons are activated during surge and tonic GnRH secretion remains to be determined.

Acknowledgments

We thank Heather Bungard and Jennifer Lydonat (West Virginia University Food Animal Research Facility) for care of animals, and Paul Harton (Histology Technician, Department of Physiology and Pharmacology, West Virginia University) for his technical assistance in sectioning tissue. We also thank Dr. Al Parlow and the National Hormone and Peptide Program for reagents used to measure LH.

This work was supported by National Institutes of Health R01 Grants HD033916 and HD017864 to (M.N.L. and R.L.G.) and P01 HD44232 (to M.N.L.), and National Science Foundation Grant 0620195 (to H.J.B.). It was also facilitated by Grant P20RR16477 from the National Center for Research Resources awarded to the West Virginia Institutional Development Award Network for Biomedical Research Excellence.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
ARC
Arcuate nucleus
AVPV
anteroventral periventricular nucleus
CIDR
controlled internal drug releasing
E2
estradiol
ER
estrogen receptor
KNDy
kisspeptin/neurokinin B/dynorphin
OVX
ovariectomized/ovariectomy
PGF2α
prostaglandin F2α
POA
preoptic area.

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