Introduction

In sheep and goats, the exposure of seasonally anestrous females to sexually mature males accelerates reproductive activity and subsequently induces out-of-season ovulation [1-5], a phenomenon called the “male effect.” The male–female interaction consists of several factors, including olfactory, visual, auditory, and tactile/behavioral cues [4,5]. Because exposing females solely to ram fleece or buck hair can induce ovulation, a pheromone released by the male possibly plays a major role in the male effect [5-9] The initial endocrine event following the reception of the male pheromone in the female is an increase in the frequency of episodic luteinizing hormone (LH) secretion. Therefore, the central target of the pheromone signal is believed to be the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator [2,4,10,11] that governs pulsatile GnRH secretion and thereby pulsatile LH secretion [12].

By recording multiple-unit activity (MUA) in the mediobasal hypothalamus, GnRH pulse generator neural activity has been successfully represented as periodic bursts of MUA (termed “MUA volleys”) in conscious animals such as monkeys [13,14], rats [15,16], and goats [17,18]. Hamada et al. demonstrated that exposure to the male pheromone immediately induced an MUA volley with an accompanying LH pulse in female Shiba goats (Capra hircus), confirming for the first time that the pheromone signal indeed activates the GnRH pulse generator [19]. Since this initial report, MUA volley induction has been used as a reliable index to assess pheromone activity in this species [10,20-22]. Shiba goats are nonseasonal breeders under natural daylight [23]. Although the male effect was initially identified as the phenomenon in anestrous females of seasonal breeders [1-5], our previous studies have shown that the induction of the MUA volley by the male pheromone can be observed independent of the season in ovariectomized (OVX) Shiba goats [10,19-22]. In this experimental representation of the male effect, timing of pheromone exposure is critical [10,19]. In this regard, a relatively constant interval of spontaneous MUA volleys in OVX goats [17,18,24] provides practical merit because timing of the next MUA volley can be anticipated, and thus timed pheromone exposure can be done between two successive MUA volleys as described below. The studies in goats unambiguously indicate that the male pheromone activates the GnRH pulse generator. However, no study has identified a specific neuronal population within the mediobasal hypothalamus responsible for MUA volley generation. Thus, the neural identity for the central target of the male pheromone remains unknown.

Human genetic studies have revealed that kisspeptin and neurokinin B (NKB) are involved in the central control mechanism of GnRH secretion [25-27]. In concert, it has been demonstrated that administration of kisspeptin or NKB (or their analogues) increases LH secretion [28-30], whereas the administration of their antagonists suppresses LH secretion [29,31-33]. A population of neurons in the arcuate nucleus (ARC) co-expresses these 2 neuropeptides in a variety of mammals, including mice [34], rats [35], sheep [36], goats [24], and monkeys [29]. Recent emerging evidence suggests that the population of kisspeptin/NKB neurons in the ARC is a likely candidate for the intrinsic source of the GnRH pulse generator [24,34,37-39]. This hypothesis is supported by findings that the active electrode that detected the MUA volley was indeed located in close proximity to kisspeptin/NKB neurons in the caudal portion of the ARC in both male [40] and female [24] goats. Therefore, it is reasonable to hypothesize that the neural signal of the male pheromone is conveyed to, and processed in, ARC kisspeptin/NKB neurons to facilitate GnRH pulse generator activity.

To address this hypothesis, the present study aimed to clarify 2 issues. First, because NKB signaling has been suggested to play a pivotal role in GnRH-pulse-generating mechanisms in kisspeptin/NKB neurons [24,38], we investigated whether NKB signaling participates in the pheromone action cascade in OVX Shiba goats. Goats implanted with an MUA recording electrode were treated with the NKB receptor antagonist SB222200 [29], and the effects of the male pheromone on the MUA volley and LH pulse were examined. Second, we investigated neural pathways involved in pheromone signal transduction to ARC kisspeptin/NKB neurons using the tract-tracing method. In general, it has been thought that pheromone signals detected by chemosensory neurons are conveyed to the medial nucleus of the amygdala (MeA). The MeA, in turn, transmits the signals directly or via the bed nucleus of the stria terminalis (BNST) to the hypothalamic nuclei, which controls behavioral and neuroendocrine outputs [41-43]. Furthermore, it has been shown that electrical stimulation or lesion of the MeA [44] modulates LH secretion in rats. Therefore, we injected an anterograde tracer, biotin dextran amine (BDA), to the MeA of OVX goats and examined using BDA histochemical analyses whether the MeA send efferent fibers to the ARC. We also evaluated whether ARC kisspeptin/NKB neurons receive projections from the MeA by using dual-labeling histochemical analyses for BDA and kisspeptin. Because pulsatile GnRH/LH release is indispensable for reproductive success [12], elucidation of neural pathways that facilitate GnRH pulse generator activity, such as the pheromone signal transduction pathway, might contribute to better understanding of the central control mechanism of reproduction in mammals.

Materials and Methods

Results

To examine the possible involvement of NKB signaling in pheromone action, goats were exposed to the pheromone in the absence or presence of the NK3R antagonist treatment. Two representative examples of MUA profiles and plasma LH concentrations are shown in Figure 1. As expected, pheromone exposure induced an MUA volley accompanied by an LH pulse within 30 s in the 5 vehicle-treated goats, resulting in a significant reduction in the intervolley interval after pheromone exposure than in the control period (21.7 ± 5.2 min vs. 31.0 ± 6.7 min, n=5, p < 0.001). After the pheromone-induced MUA volley, MUA volleys occurred at regular intervals, similar to what was observed in the control period (Figure S1). In contrast, pheromone exposure failed to induce an MUA volley and an LH pulse in the goats treated with SB222200. Compared with the control period, the intervolley interval after pheromone exposure was significantly larger (48.0 ± 6.2 min vs. 29.3 ± 5.0 min, n=5, p < 0.001). There were no apparent changes in the basal MUA profiles after injection of SB222200.

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Figure 1. Multiple-unit activity (MUA) profiles and plasma luteinizing hormone concentrations in two representative ovariectomized goats.

Goats were exposed to the male pheromone in the absence (upper panels) or presence (lower panels) of the NK3R antagonist SB222200. The timing of pheromone exposure is indicated by an arrow and dotted line. Vehicle (open arrowheads) or SB222200 (closed arrowheads) were injected intravenously twice (at the first and fourth blood sampling points) after the preceding MUA volley.

https://doi.org/10.1371/journal.pone.0081017.g001

Figure 2 shows photomicrographs of the injection site in 3 OVX goats, and a goat brain atlas showing the MeA with schematic drawings of the injection site. Densely packed BDA-positive products characterized the injection site, which was elliptical or round in shape, with an approximate diameter of 1.5–3 mm. Of the 6 goats used in the tract-tracing study, 2 had the injection site confined to the MeA (Figure 2A, 2B, and 2E), and 1 had the injection site located in the ventrolateral region of the MeA, with some extension into the adjacent basal amygdala and posterior medial cortical amygdala (Figure 2C and 2E). A higher magnification shows a number of cells that had taken up BDA at the injection site (Figure 2D). In the remaining 3 goats, the injection site did not contain the MeA.

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Figure 2. Biotin dextran amine (BDA) injection into the medial nucleus of the amygdala (MeA).

Photomicrographs of sections stained for BDA show the injection site in the MeA in 3 ovariectomized goats (A–C). The injection site is characterized by densely packed BDA-positive signals. D, High magnification of the squared area in B. Open arrowheads indicate cells that took up BDA. E, The goat brain atlas, including the amygdaloid complex and the injection site. Shaded areas (A–C) correspond to respective photomicrographs (A–C). Amygdaloid complex: BA, basal nucleus; CeA, central nucleus; MeA, medial nucleus; LA, lateral nucleus; PLCo, posterolateral cortical nucleus; PMCo, posteromedial cortical nucleus; AHA, anterior hypothalamic area. Other structures: BNST, bed nucleus of the stria terminalis; En, endopiriform cortex; PVH, paraventricular nucleus of the hypothalamus; PYR, piriform cortex; PUT, putamen; SON, supraoptic nucleus; ac, anterior commissure; fx, fornix; LV, lateral ventricle; ME, median eminence; ot, optic tract; stm, stria medullaris of the thalamus; 3V, third ventricle. Scale bar: A = 1 mm for A-C; D = 50 µm.

https://doi.org/10.1371/journal.pone.0081017.g002

Photomicrographs of BDA-labeled fibers in the hypothalamus and BNST are presented in Figure 3. The distribution of labeled fibers in a goat in which the injection was confined to the MeA (Figure 2B) is schematically shown in Figure 4. Efferent fibers of the MeA formed a dense plexus in the BNST and then coursed ventrally and medially to the hypothalamus (Figure 3A and 4A). Some efferent fibers of the MeA, perhaps not passing the BNST, coursed ventrally in areas over the optic tract (Figure 3B) and reached rostrally to areas ventral to the anterior hypothalamic area (Figure 4A) as well as caudally to the lateral hypothalamic area (LHA). They merged with descending labeled fibers from the BNST in the lateral region of the ARC. Labeled fibers were observed in several hypothalamic nuclei and were abundant in the ventromedial nucleus of the hypothalamus (VMH) and LHA. In particular, they formed a dense plexus in the VMH (Figure 3C). The ARC also contained a substantial number of labeled fibers (Figure 3D and 3E). Although labeled fibers were observed throughout the rostro-caudal extent of the nucleus, their density was markedly low in the caudal region (Figure 4B–D). Within the ARC, labeled fibers were predominantly distributed at its dorsal and lateral aspects. They were branched and possessed varicosities along with the axon and apparent terminal buttons (Figure 3F).

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Figure 3. Photomicrographs of biotin dextran amine-labeled fibers.

A, Labeled fibers forming a dense plexus in the bed nucleus of the stria terminalis. B, Labeled fibers coursing areas over the optic tract. C, Labeled fibers forming a dense plexus in the ventromedial nucleus of the hypothalamus. D, E, Labeled fibers distributed in the rostral (D) and middle region (E) of the arcuate nucleus. F, High magnification of the area indicated in E. Arrowheads indicate apparent terminal buttons. ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; LHA; lateral hypothalamic area; VMH, ventromedial nucleus of the hypothalamus; fx, fornix; LV, lateral ventricle; ot, optic tract; pt, par tuberalis; 3V, third ventricle. Scale bar: A = 300 µm; B-E =150 µm; F= 20 µm.

https://doi.org/10.1371/journal.pone.0081017.g003

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Figure 4. Schematic drawings of the distribution of labeled fibers.

A, Frontal illustration of areas immediately rostral to the rostral edge of the arcuate nucleus (ARC). B–D, Frontal illustrations of the mediobasal hypothalamus containing the rostral (B), middle (C), and caudal (D) regions of the ARC. LHA; lateral hypothalamic area; ARC, arcuate nucleus; BNST, bed nucleus of the stria terminalis; PVH, paraventricular nucleus of the hypothalamus; SON, supraoptic nucleus; RCh, retrochiasmatic are; VMH, ventromedial nucleus of the hypothalamus; fx, fornix; LV, lateral ventricle; ME, median eminence; ot, optic tract; pt, par tuberalis; stm, stria medullaris of the thalamus; 3V, third ventricle. Scale bar: A = 1 mm; B = 500 µm for B-D.

https://doi.org/10.1371/journal.pone.0081017.g004

The abovementioned distribution pattern of BDA-labeled fibers was similar among the goats that received BDA injection within the MeA (Figure 2). The density of labeled fibers in the ARC was relatively lower in the goat with the injection site located at the ventrolateral part of the MeA (Figure 2C) compared with that in the other 2 goats. Although some labeled fibers were occasionally observed in the contralateral side of the injection site, BDA-positive fibers were distributed predominantly in the ipsilateral side. As observed in previous studies using this tracer [49,50], retrogradely labeled cells were occasionally observed in all regions examined. In goats that received the BDA injection outside of the MeA (n = 3), labeled fibers were scarcely distributed in the hypothalamus, and few or no labeled fibers were observed in the ARC.

To examine whether ARC kisspeptin neurons are connected with the MeA, dual-labeling histochemical analyses for BDA and kisspeptin were performed. Fluorescence microscopic observations indicated that several BDA-positive fibers run and terminate in close vicinity with kisspeptin-immunoreactive (-ir) neurons in the ARC (Figure 5A). It appeared that some appose kisspeptin-ir neurons (Figure 5B). Such occurrences were observed approximately 0–3 instances per slide. However, based on fluorescence microscopic observation, it was unclear whether they represented direct contact between BDA-positive fibers and kisspeptin-ir neurons. Therefore, several slides were analyzed further using confocal microscopy. These analyses revealed that some BDA fibers actually appose kisspeptin-ir neural processes (perhaps dendrites) (Figure 5C and 5D) or kisspeptin-ir cell bodies (Figure 5E and 5F). The occurrence of apposition was, however, relatively low.

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Figure 5. Photomicrographs of dual-labeling histochemical analyses for biotin dextran amine (BDA) and kisspeptin in the arcuate nucleus (ARC).

A, B, Fluorescence microscopy images of BDA-labeled fibers (green) and kisspeptin-immunoreactive (-ir) neurons (red). C–F, Confocal microscopy images of BDA-labeled fibers (green) and kisspeptin-ir neurons (red). C, E, Stacked images of 15 serial confocal planes. D, F, A single 1-µm thick plane of pictures in C and E, respectively. Arrows indicate kisspeptin-ir neurons. Open arrowheads indicate BDA-labeled fibers running in close proximity to kisspeptin-ir neurons. Closed arrowheads indicate direct apposition of BDA-labeled fibers with kisspeptin-ir neurons. Scale bar: A = 100 µm; B = 50 µm; C = 40 µm for C and D; E = 40 µm for E and F.

https://doi.org/10.1371/journal.pone.0081017.g005

Discussion

Although it has long been accepted that the central target of the male pheromone signal is the putative hypothalamic GnRH pulse generator [2,4,10,11], the precise neural mechanisms underlying pheromone action remained unclear. In the present study, we demonstrated in goats that pheromone action on GnRH pulse generator activity depends on entirely NKB signaling, and ARC kisspeptin neurons receive projections from the MeA. NKB signaling is suggested to play a pivotal role in the GnRH pulse generation by ARC kisspeptin neurons [24,38,39,46,51]. Moreover, the MeA is considered to be a key neural substrate that relays pheromone signals to target nuclei in the hypothalamus [41-43,52]. Considering these findings, the present results strongly suggest that the male pheromone signal is transmitted to, and processed in, a subset of ARC kisspeptin/NKB neurons, triggering pulsatile GnRH/LH secretion.

Consistent with previous studies [10,19-22], an MUA volley was induced within 30 s after exposure to the pheromone in all vehicle-treated control goats in this study. However, when goats were treated with the NK3R antagonist SB222200, neither the MUA volley nor the LH pulse was observed upon pheromone exposure (Figure 1). These results demonstrate that NK3R antagonists negate the action of the male pheromone on the GnRH pulse generator, thereby suppressing pulsatile LH secretion in goats. Although NK3Rs are distributed in the central as well as the peripheral nervous system [53], the action of peripherally administered SB222200 on the male effect might be exerted centrally as it affected MUA volley induction. There are 2 possible sites of action for the antagonist in the brain. One is the ARC and the other is the retrochiasmatic area (RCh). It has been demonstrated in sheep that a subset of neurons in the RCh contain NK3R [54], and local administration of an NK3R agonist into the RCh dramatically increases LH secretion [55]. Therefore, NK3R-containing RCh neurons may be involved in the control of GnRH secretion and, thus, in the stimulatory action of the pheromone on GnRH/LH release. However, this seems unlikely because activation of NKB signaling in the RCh results in a surge-like LH release [55], whereas the pheromone induces pulsatile small amplitude LH secretion. It is more likely that SB222200 negates pheromone action by blocking NKB signaling in ARC kisspeptin neurons. A majority of ARC kisspeptin/NKB neurons co-expresses NK3R in sheep [54] and goats [56]. The observation that central administration of NKB activated approximately 70% of ARC kisspeptin/NKB neurons and induced pulse-like LH secretion in seasonally anestrous ewes [30] suggests that NKB signaling in ARC kisspeptin/NKB neurons is associated with pulsatile GnRH/LH secretion. Moreover, the electrode monitoring MUA was targeted to ARC kisspeptin/NKB neurons in the present and previous [10] studies, where it was unambiguously demonstrated that the pheromone induces the MUA volley.

In the present study, the intervolley interval after SB222200 administration was significantly prolonged as compared with that in the control period. This result indicates that the antagonist suppressed the pheromone-induced as well as the spontaneously occurring MUA volleys. Therefore, the effect of the antagonist might not be specific for pheromone action, rather may be an overall suppression of GnRH pulse generator activity itself. Recent studies in rats [51] and goats [46] have demonstrated that ARC kisspeptin/NKB neurons are interconnected and suggest that NKB signaling may play a role in synchronized bursting activities in such a neural network. In this context, it can be assumed that the antagonist prevented synchronized bursting among ARC kisspeptin/NKB neurons, thereby negating pheromone action and suppressing the occurrence of spontaneous MUA volleys. A recent study showed that infusion of a kisspeptin receptor antagonist [31] into the lateral ventricle completely blocked the effect of ram exposure on LH secretion in seasonally anestrous ewes [57]. Therefore, it is likely that kisspeptin signaling, as well as NKB signaling, plays critical roles in the central mechanism of the male effect, in which the pheromone signal is translated to pulsatile GnRH/LH secretion.

The present anterograde tracer study demonstrated that the MeA sends projections to several hypothalamic nuclei, including the ARC (Figure 4). The pathway and distribution of the efferent fibers of the MeA observed in goats are comparable to those demonstrated in rats [58,59] and hamsters [60], suggesting that these neural pathways are highly conserved among mammals. Efferent fibers of the MeA were branched and possessed varicosities and terminal buttons in the ARC (Figure 3F), indicating that the ARC is the terminal field for MeA efferent fibers. Moreover, dual-labeling histochemical analyses revealed that kisspeptin neurons in the ARC received apposition of fibers originating from the MeA (Figure 5D and 5F). These results indicate that the MeA sends projections to ARC kisspeptin/NKB neurons.

Although the results of the tracer study clearly demonstrate the presence of a neural connection between the MeA and ARC kisspeptin/NKB neurons, the anatomical data do not provide direct insights regarding whether such a neural pathway is indeed involved in male pheromone signal transduction. However, several lines of evidence suggest that this may be the case. First, all putative pheromone receptors, V1Rs, identified thus far in goats are expressed in both vomeronasal and olfactory sensory neurons [61,62], and the MeA receives inputs from these 2 types of sensory neurons via the accessory and main olfactory bulbs, respectively [63,64]. Second, the MeA is proposed to be a key nucleus that relays pheromone signals to target nuclei in the hypothalamus [41-43]. Third, exposure of seasonally anestrous ewes to rams results in the induction of c-Fos, a marker of neuronal activation, in MeA neurons [65] as well as in ARC kisspeptin/NKB neurons [57]. Finally, the male effect in goats and sheep depends on NKB (this study) and kisspeptin [57] signaling, both of which are associated with ARC kisspeptin/NKB neurons. Therefore, it is possible that the MeA relays the male pheromone signal to a subset of ARC kisspeptin/NKB neurons, where it is translated to neural activity that elicits pulsatile GnRH release through NKB and kisspeptin signaling.

Although a substantial number of MeA efferent fibers terminate in the ARC, the direct apposition of these fibers on kisspeptin cell bodies was observed in a few instances. This raises the possibility that other neurons in the ARC could receive the pheromone signal from the MeA and relay the signal to ARC kisspeptin/NKB neurons. ARC kisspeptin neurons are known to receive inputs from neuropeptide Y (NPY), proopiomelanocortin, and dopamine neurons in sheep [66,67]. The cell bodies of these neurotransmitters are also known to reside in the ARC. Therefore, the pheromone action may be mediated by either of these neurotransmitters. However, this is unlikely because it has been shown that NPY suppresses GnRH pulse generator activity in goats [18], and dopamine acts to inhibit LH secretion in anestrous ewes [67]. Moreover, although there is evidence that central administration of a melanocortin receptor agonist slightly stimulates GnRH pulse generator activity, this effect was observed 10–15 min after administration [68], which is in contrast to the rapid action of the pheromone (within 1 min). Thus, whether other neurons, in addition to kisspeptin neurons, are involved in pheromone action remains unclear. Alternatively, pheromone induction of the MUA volley and LH pulse may be mediated by a small subset of ARC kisspeptin neurons. ARC kisspeptin/NKB neurons are interconnected with each other to form a neuronal circuit [46,51]. Studies have suggested that the initial activation aroused in a subset of kisspeptin neurons would be amplified and propagated among cells within this circuit to evoke synchronized bursting of kisspeptin neurons [24,34,38]. In this case, ARC kisspeptin/NKB neurons may not necessarily receive rich projections from the MeA, and this may explain why apposition of the MeA/BNST projections on kisspeptin neurons was observed only in a few instances.

In conclusion, we demonstrated in the present study that the MUA volley and LH responses to male pheromone are thoroughly suppressed by administration of an NKB receptor antagonist. We also demonstrated that a subset of ARC kisspeptin/NKB neurons receive efferent projections from the MeA in goats. These results suggest that the chemosensory signal of the male pheromone is conveyed via the MeA to ARC kisspeptin/NKB neurons, where the signal stimulates pulse generator activity through an NKB signaling-mediated mechanism.

Supporting Information

Acknowledgments

We thank Drs. T. Ohtaki and H. Matsumoto (Takeda Pharmaceutical Co.) for providing the anti-rat kisspeptin antibody (Takeda #254). The authors wish to thank Ms. Y. Sakairi for her valuable technical assistance throughout the study.