Low Degree of Overlap Between Kisspeptin, Neurokinin B, and Dynorphin Immunoreactivities in the Infundibular Nucleus of Young Male Human Subjects Challenges the KNDy Neuron Concept

Erik Hrabovszky; Máté T Sipos; Csilla S Molnár; Philippe Ciofi; Beáta Á Borsay; Péter Gergely; László Herczeg; Stephen R Bloom; Mohammad A Ghatei; Waljit S Dhillo; Zsolt Liposits

doi:10.1210/en.2012-1545

. 2012 Aug 17;153(10):4978–4989. doi: 10.1210/en.2012-1545

Low Degree of Overlap Between Kisspeptin, Neurokinin B, and Dynorphin Immunoreactivities in the Infundibular Nucleus of Young Male Human Subjects Challenges the KNDy Neuron Concept

Erik Hrabovszky ^1,^✉, Máté T Sipos ¹, Csilla S Molnár ¹, Philippe Ciofi ¹, Beáta Á Borsay ¹, Péter Gergely ¹, László Herczeg ¹, Stephen R Bloom ¹, Mohammad A Ghatei ¹, Waljit S Dhillo ¹, Zsolt Liposits ¹

PMCID: PMC3512020 PMID: 22903610

Abstract

Previous immunohistochemical and in situ hybridization studies of sheep, goats, and rodents indicated that kisspeptin (KP), neurokinin B (NKB), and dynorphin A (DYN) are extensively colocalized in the hypothalamic arcuate nucleus, thus providing a basis for the KP/NKB/DYN (KNDy) neuron concept; in both sexes, KNDy neuropeptides have been implicated in the generation of GnRH neurosecretory pulses and in the negative feedback effects of sexual steroids to the reproductive axis. To test the validity and limitations of the KNDy neuron concept in the human, we carried out the comparative immunohistochemical analysis of the three neuropeptides in the infundibular nucleus (Inf; also known as arcuate nucleus) and stalk of young male human individuals (<37 yr). Results of quantitative immunohistochemical experiments established that the regional densities of NKB immunoreactive (IR) perikarya and fibers, and the incidence of afferent contacts they formed onto GnRH neurons, were about 5 times as high as those of the KP-IR elements. Dual-immunofluorescent studies confirmed that considerable subsets of the NKB-IR and KP-IR cell bodies and fibers are separate, and only about 33% of NKB-IR perikarya and 75% of KP-IR perikarya were dual labeled. Furthermore, very few DYN-IR cell bodies could be visualized in the Inf. DYN-IR fibers were also rare and, with few exceptions, distinct from the KP-IR fibers. The abundance and colocalization patterns of the three immunoreactivities showed similar trends in the infundibular stalk around portal blood vessels. Together these results indicate that most NKB neurons in the Inf do not synthesize detectable amounts of KP and DYN in young male human individuals. These data call for a critical use of the KNDy neuron terminology when referring to the putative pulse generator system of the mediobasal hypothalamus. We conclude that the functional importance of these three neuropeptides in reproductive regulation considerably varies among species, between sexes, and at different ages.

Information accumulated from immunohistochemical and in situ hybridization studies has recently formed the basis for the kisspeptin/neurokinin B/dynorphin A (KNDy) neuron concept and terminology (1–4). As shown first for the sheep (5), many of these neurons with cell bodies located in the hypothalamic arcuate nucleus [ARC; called infundibular nucleus in humans (Inf)] cosynthesize kisspeptin (KP), neurokinin B (NKB), and dynorphin A (DYN). They have been implicated in negative sex steroid feedback to GnRH neurons (5–7) and proposed to also serve as pacemakers for the GnRH neurosecretory pulses (3, 4, 8, 9). Recent models of the GnRH pulse generator (3, 4, 8) suggest that KNDy neurons communicate with one another via NKB and its receptor, NK3, and possibly, also DYN and its receptor, KOR. In ovariectomized goats, central NKB increases and DYN decreases the frequencies of multiunit activity volleys and LH secretory pulses (8). Pulse generator cells, in turn, appear to communicate with GnRH neurons primarily via KP/KP receptor (KISS1R) signaling. GnRH neurons express KISS1R (10–12), and the majority of GnRH neurosecretory pulses show temporal association with KP pulses in the median eminence of monkeys (13).

The general consensus that KP, NKB, DYN, NK3, and KOR are expressed by the same neurons relies on combined neuroanatomical data from sheep (1, 5), rats (14), mice (3, 4, 15), goats (8), monkeys (16), and humans (17, 18). However, closer analysis of these reports, in retrospect, reveals that neuropeptide and receptor colocalizations are often only partial and also variable in the different studies, species, sexes, and age groups. Notably, in our recent immunohistochemical study of aged human individuals, we have detected robust sex differences in the abundance of KP-immunoreactive (IR) (17, 18) and NKB-IR (18) neuronal elements in the Inf. These studies have also revealed that the incidences of NKB-IR cell bodies, fibers, and appositions onto GnRH neurons exceed severalfold those of KP-IR elements, with particularly robust differences in males (18). These results suggested that NKB-IR neurons and their fibers are partly distinct from the KP-IR elements in these human models, thus challenging the universal validity of the KNDy neuron concept. Moreover, in triple-immunofluorescent studies of aged human males, GnRH neurons tended to receive primarily single-labeled afferent inputs from these peptidergic systems, with the KP/NKB double-labeled axons representing only 10.2% of all KP-IR afferents and the NKB/KP double-labeled axons about 8.8% of all NKB-IR afferents (18). From the above findings and preliminary observations indicating that KP immunolabeling is even weaker in young than in aged men, we predicted that the degree of overlap between the KNDy neuropeptides is much lower in young male humans than was suggested earlier for female sheep (1, 5), goats (8), or mice (3).

In the present study, we investigated the universal validity of the KNDy neuron concept via the parallel immunohistochemical analysis of NKB-, KP-, and DYN immunoreactivities in the Inf and the infundibular stalk (InfS) of young men. Specifically, 1) we compared the immunoreactive perikaryon and fiber densities in the Inf and the InfS, 2) we addressed the colocalization of KP with NKB and KP with DYN in perikarya and fibers, and finally, 3) we compared quantitatively the incidences of NKB-IR vs. KP-IR afferent contacts onto GnRH-IR neurons.

Materials and Methods

Human subjects

Human hypothalamic samples were obtained from autopsies at the Forensic Medicine Department of the University of Debrecen with permission from the Regional Committee of Science and Research Ethics of the University of Debrecen (DEOEC RKEB/IKEB: 3183-2010). Selection criteria included sudden causes of death, lack of a history of neurological and endocrine disorders, and postmortem delay less than 36 h. Tissue specimens from six young male individuals (aged 21–37 yr) were used.

Section preparation

After dissection, the hypothalamic tissue blocks were rinsed with running tap water and then immersion fixed with 4% formaldehyde in 0.1 m PBS (pH 7.4) for 7–14 d. After fixation, the blocks were trimmed in a way to include the optic chiasma rostrally, the mammillary bodies caudally and the anterior commissure dorsally (17–19). Bilateral sagittal cuts were placed 2 cm lateral from the midline. The blocks were bisected into right and left halves and then infiltrated with 20% sucrose for 5 d at 4 C. The right hemihypothalami were sectioned coronally at 30 μm with a Leica SM 2000R freezing microtome (Leica Microsystems, Nussloch GmbH, Germany). All experiments were performed on every 24th hemihypothalamic section from each subject.

Pretreatments

The tissues were permeabilized and endogenous peroxidase activity reduced using a mixture of 0.2% Triton X-100 and 0.5% H₂O₂ in PBS for 30 min. Antigen epitopes were unmasked by incubating sections in 0.1 m citrate buffer (pH 6.0) at 80 C for 30 min (18). Dual-immunofluorescent experiments also used a Sudan black pretreatment against autofluorescence (17, 18).

Immunohistochemical detection of KP

To detect KP immunoreactivity, sections were incubated in a sheep polyclonal antiserum against human kisspeptin-54 (GQ2; 1:200,000). This antiserum recognizes human KP-54, KP-14, and KP-10, exhibits less than 0.01% cross-reactivity in vitro with other related human RF amide peptides (20), and was used successfully in previous immunohistochemical experiments on primate hypothalami (16–18, 21). Incubation in the primary antibodies for 48 h at 4 C was followed by biotinylated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:500) and the ABC Elite reagent (Vector Laboratories, Burlingame, CA; 1:1000) for 60 min each. The peroxidase reaction was visualized with nickel-intensified diaminobenzidine chromogen (19) and then postintensified with silver-gold (22).

Immunohistochemical detection of NKB and DYN

Another two parallel series of sections were used to visualize NKB and DYN, respectively. NKB neurons were detected with rabbit polyclonal antibodies against the C-terminal 28 amino acids of human pro-NKB (IS-682, P. Ciofi; 1:100,000) (17), whereas DYN neurons were labeled with rabbit polyclonal antibodies against amino acids 1–17 of porcine (and human) DYN (T-4268; Peninsula Laboratories; San Carlos, CA; 1:100,000). The primary antibodies were reacted with biotinylated antirabbit IgG (Jackson ImmunoResearch Laboratories; 1:500; 1 h) and then ABC Elite reagent (1:1000; 1 h). The peroxidase signal was developed with silver-gold-intensified nickel-diaminobenzidine. For control purposes, the mapping studies of DYN were replicated using a previously characterized second antiserum (IS-35; 1:200,000) against a different prodynorphin-derived peptide, dynorphin B (23).

Dual-immunoperoxidase detection of KP and GnRH or NKB and GnRH

Two section series were processed for the immunohistochemical detection of KP and NKB, respectively, as above. Then they were processed to detect GnRH using a guinea pig primary antiserum (no. 1018; 1:5000) (18), which was reacted with biotinylated antiguinea pig IgG (Jackson ImmunoResearch Laboratories; 1:500; 1 h) and ABC Elite solution (Vector Laboratories; 1:1000; 1 h). The peroxidase signal was visualized with brown diaminobenzidine.

Fluorescent immunohistochemistry

Other series of sections were processed for the dual-immunofluorescent studies of the colocalization between NKB and KP or DYN and KP. Incubation in a cocktail of primary antibodies (rabbit anti-NKB, 1:1000; and sheep anti-KP, 1:1000; or rabbit anti-DYN, 1:1000; and sheep anti-KP, 1:1000; 48 h; 4 C) was followed by a cocktail of fluorochrom-conjugated secondary antibodies [Jackson ImmunoResearch; antirabbit-fluorescein isothiocyanate (FITC), 1:250; antisheep-Cy3, 1:1000] for 5 h at room temperature.

To maximize sensitivity, other dual-immunofluorescent studies used tyramide signal amplification. In these experiments KP was detected first using sequential incubations in KP antibodies (1:30,000; 48 h; 4 C), biotinylated antigoat IgG (Jackson ImmunoResearch Laboratories; 1:500; 1 h), the ABC Elite reagent (Vector Laboratories; 1:1,000; 1 h), biotin tyramide working solution [1:1,000, in 0.05 m Tris-HCl buffer (pH 7.6), containing 0.003% H₂O₂; 30 min] (24), and finally, avidin-Cy-3 (Jackson ImmunoResearch; 1:1,000; 1 h). Then the sections were treated for 30 min with 0.5% H₂O₂ and 0.1% sodium azide in PBS to inactivate horseradish peroxidase. To detect NKB or DYN, the rabbit primary antibodies were used at 1:50,000 (48 h; 4 C) and reacted with antirabbit-peroxidase (Jackson ImmunoResearch; 1:500; 1 h). Then FITC-tyramide (24) [diluted 1:500 with 0.05 m Tris-HCl buffer (pH 7.6), containing 0.003% H₂O₂; 30 min] was deposited on the peroxidase sites. Control experiments included the omission of the NKB and DYN primary antibodies. Lack of FITC labeling in these control sections indicated that no FITC-tyramide deposition is caused by residual peroxidase activity on KP-IR sites.

Section mounting and coverslipping

After section mounting, immunoperoxidase-labeled sections were coverslipped with DPX (Fluka Chemie, Buchs, Switzerland) and immunofluorescent specimens with Mowiol.

Digital photography

The light and fluorescent microscopic images were scanned with an AxioCam MRc 5 digital camera mounted on a Zeiss AxioImager M1 microscope using the AxioVision 4.6 software (Carl Zeiss, Göttingen, Germany). Confocal images were prepared with an inverted Nikon Eclipse Ti-E microscope equipped with an A1R confocal system (Nikon, Vienna, Austria). The digital images were processed with the Adobe Photoshop CS software (Adobe Systems, San José, CA) at a 300-dpi resolution. Quantitative data were expressed as mean ± sem and statistical comparisons used one-way ANOVA.

Experiment 1. Studies of the incidences and overlaps of IR cell bodies in the Inf

The number of immunoreactive cell bodies was counted at 100 × magnification within a 0.25-mm² counting area with the aid of a 5 × 5 ocular grid, as described previously (17, 18). Each subject was characterized by the maximal number of immunoreactive perikarya in this counting area (determined from two to six sections.)

The overlaps between NKB and KP immunoreactivities and between DYN and KP immunoreactivities were first assessed in confocal images of dual-immunofluorescent specimens. The percentages of single-labeled and double-labeled NKB-IR and KP-IR perikarya were also determined quantitatively from the specimens in which the tyramide signal amplification was used, using one to three representative confocal images per subject.

Experiment 2. Studies of the regional abundance of IR fibers in the Inf

The regional density of immunoreactive fibers was determined as described recently (18). First, digital images were taken from the bulk of KP-IR and NKB-IR neurons in the Inf. The files were opened with the Adobe Photoshop CS software. The immunolabeled cell bodies were erased (eraser tool) from the photomicrographs. The remaining images were compiled into TIF files and opened with the ImageJ software (public domain at http://rsbweb.nih.gov/ij/download.html). The regional fiber density in each photograph was defined as the area occupied by immunoreactive fibers/total area. For each subject, the mean fiber density was derived from one to three digital images. The overlap between NKB-IR and KP-IR axons or DYN-IR and KP-IR axons was also studied qualitatively in confocal images of dual-immunofluorescent specimens.

Experiment 3. Studies of immunoreactive fibers in the InfS

Projections of NKB-IR, KP-IR, DYN-IR, and GnRH-IR axons around the portal blood vessels of the InfS were analyzed in this experiment. Based on previous immunohistochemical results in the median eminence of different species (16, 23), we assumed that fibers containing KNDy peptides around the portal vasculature arise mostly from the ARC/Inf. First, sections labeled with peroxidase-based immunohistochemistry were used to study the relationship of fibers with the superficial and deep capillary plexuses of the human postinfundibular eminence (25). Then the extents of overlap between NKB and KP immunoreactivities and between DYN and KP immunoreactivities were assessed from dual-immunofluorescent specimens.

Experiment 4. Studies of the incidences of KP-IR and NKB-IR appositions onto GnRH-IR neurons of the Inf

Dual-immunoperoxidase labeled sections were selected (one to two from each individual) to determine the number of axonal contacts along the outlines of GnRH-IR cell bodies and dendrites. Counting of the appositions was carried out using a ×63 oil-immersion objective and contacts defined using stringent criteria (18, 26, 27). For each subject, the mean number of contacts per GnRH soma and 100 μm GnRH dendrite were calculated (18).

Results

The comparative analysis of NKB, KP, and DYN immunoreactivities in immunoperoxidase-labeled sections of the Inf (Fig. 1) and the InfS (Fig. 2) revealed strikingly different labeling intensities for KNDy neuropeptides. In general, NKB-IR elements showed much higher abundance than KP-IR elements. DYN immunoreactivity, both in perikarya and fibers, was relatively sparse and weak.

Experiment 1. Incidence of IR perikarya in the Inf

In peroxidase-based immunohistochemistry, many NKB-IR perikarya were identified in the Inf (Fig. 1, A and B). KP-IR cell bodies occurred in much lower numbers in neighboring sections (Fig. 1, C and D). Quantitative analysis showed that the density of KP neurons was about 5 times lower than that of NKB-IR perikarya (Fig. 5; P = 0.01 by ANOVA). DYN-IR perikarya were either entirely absent in some subjects (Fig. 1, E and F) or extremely rare in the Inf of others, preventing quantitative studies. In contrast, the supraoptic nucleus contained many labeled perikarya (Fig. 1G), making it unlikely that the low DYN signal in the Inf reflects technical limitations.

Fig. 5. — Regional abundance of NKB-IR and KP-IR neuronal perikarya in the Inf of young male humans. The maximal number of immunoreactive cell bodies per 0.25 mm² counting frame (one to six per subject) was determined with the aid of an ocular frame and used as the index of the density of labeled perikarya. Results show that in young male human individuals, the mean incidence of NKB-IR cell bodies is about 5 times as high as the incidence of KP-IR cell bodies. *, P < 0.05 by ANOVA.

A surprising partial segregation of NKB-IR and KP-IR perikarya was revealed in dual-immunofluorescent specimens (Fig. 3, A and B). Tyramide signal amplification was crucial for sensitive detection of NKB/KP dual-labeled cell bodies which represented only 32.9 ± 4.7% of the NKB-IR and 75.2 ± 6.6% of the KP-IR perikarya (Fig. 3, A and B). Tyramide signal amplification was capable of visualizing only a few DYN-IR perikarya (not shown).

Experiment 2. Abundance of IR fibers in the Inf

The incidence of immunolabeled fibers in the Inf followed a similar trend as shown by labeled perikarya. The most frequently encountered phenotype was, again, IR for NKB. These axons established many appositions (Fig. 1B) to NKB-IR cell bodies and their dendritic processes. Quantitative analysis of the area covered by immunohistochemical signal established that the mean incidence of NKB-IR fibers was about 5 times as high as that of KP-IR fibers (Fig. 6; P = 0.0001 by ANOVA). DYN-IR fibers were also detectable in the Inf, although less frequently than either NKB-IR or KP-IR axons (Fig. 1, E and F).

Fig. 6. — Density of NKB-IR and KP-IR fibers in the Inf of young men. The area covered by immunoreactive fibers (divided by the total area analyzed) was determined with the ImageJ software in digital photographs of the Inf and used as a fiber density measure. Areas that were occupied by labeled cell bodies and their thick proximal dendrites were deleted from the photographs with the eraser tool of the Adobe Photoshop software and thus excluded from the analysis. The density of NKB-IR fibers (expressed in arbitrary units) is 5.4 times as high as the density of KP-IR fibers. *, P < 0.0005 by ANOVA.

In immunofluorescent specimens, many NKB-IR fibers without KP immunolabeling as well as KP-IR fibers without NKB labeling could be seen in the Inf, in addition to dual-labeled axons (Fig. 3, A and B). DYN-IR fibers showed a high intensity of labeling only if the tyramide signal amplification approach was also used. Most of them were distinct from KP-IR axons, although dual-labeled KP/DYN-IR fibers occasionally occurred (Fig. 3, F and G). Similarly, the majority of KP-IR fibers were also devoid of dynorphin B immunoreactivity in the Inf and the InfS (Fig. 3, I and J), whereas this second antiserum also performed well in regions rich in DYN fibers, including the ventromedial nucleus (Fig. 3K).

Experiment 3. Abundance of immunoreactive fibers in the InfS

The InfS was associated with the superficial and the deep capillary plexuses of the postinfundibular eminence (25). Both were abundantly innervated by GnRH-IR axons (Fig. 2, A–F, brown color), suggesting they contribute to the GnRH supply of adenohypophysial gonadotropes. The relative abundance of the different types of labeled fibers around the two capillary plexuses matched what was seen in the Inf. Accordingly, portal blood vessels were surrounded by dense networks of NKB-IR fibers (Figs. 2, A–C) and innervated only moderately by KP-IR fibers (Figs. 2, D–F). Very few DYN-IR fibers occurred in the proximity of the portal capillaries (Figs. 2, G, I, and J). This low level of DYN signal did not reflect a technical limitation, considering that the magnocellular neurosecretory tract was immunolabeled heavily in the same sections (Fig. 2H).

The analysis of immunofluorescent specimens confirmed that NKB dominates over KP around the portal vasculature and NKB-IR fibers often lack KP labeling (Fig. 3, C–E). Similarly to the Inf, the InfS contained both single-labeled and double-labeled KP-IR fibers (Figs. 3, C–E). In sections dual labeled for KP and DYN, labeled fibers were mostly distinct, although rare colocalization cases were also detectable (Fig. 3H).

Experiment 4. Frequency of KP-IR and NKB-IR appositions onto GnRH-IR neurons

Sections double labeled with the silver-gold-intensified nickel-diaminobenzidine and diaminobenzidine chromogens were used to obtain quantitative estimates about NKB-IR and KP-IR inputs to GnRH-IR neurons. Microscopic analysis confirmed that NKB-IR (18) and KP-IR (17, 18) axons provide axosomatic and axodendritic inputs to GnRH neurons in the Inf (Fig. 4). Quantitative analysis (Fig. 7) established that GnRH-IR perikarya and dendrites, respectively, received 6 and 5 times heavier NKB-IR (Fig. 4A) than KP-IR (Fig. 4B) innervation (GnRH perikarya: P = 0.004; GnRH dendrites: P = 0.005, by ANOVA).

Fig. 4. — Different incidences of NKB-IR and KP-IR afferent contacts onto GnRH-IR neurons. NKB-IR axons (*black color* in A) show a much higher abundance in the Inf and establish considerably more axosomatic and axodendritic juxtapositions (*arrows*) onto GnRH-IR neurons (*brown color*) than do KP-IR axons (*black color* in B). For quantification of these results, see Fig. 7. *Scale bar*, 20 μm.

Fig. 7. — Incidences of NKB-IR and KP-IR contacts onto GnRH-IR neurons in the Inf in young men. High-power light microscopic analysis of sections, dual immunolabeled with the combined use of silver-gold-intensified nickel-diaminobenzidine and diaminobenzidine chromogens, was carried out to determine the relative incidences of NKB-IR and KP-IR neuronal appositions to the somata (*left columns*) and the dendrites (*right columns*) of GnRH-IR neurons. The counts were obtained from all GnRH-IR cell bodies and dendrites identified in one to three infundibular sections of each individual. The number of NKB-IR contacts is about 6 times as high on the somatic and 5 times as high on the dendritic compartment of GnRH neurons as those established by KP-IR axons. * P < 0.01 by ANOVA.

Discussion

Immunohistochemical results of this study provide evidence for limitations of the KNDy neuron terminology and concept. Specifically, our observations indicate that in young male humans the majority of NKB-IR neurons in the Inf, their processes and contacts onto GnRH neurons do not contain detectable amounts of KP immunoreactivity. Furthermore, KP-IR neuronal elements without NKB labeling also occur frequently in addition to NKB/KP dual-phenotype structures. Finally, we observed that most KP-IR neurons and fibers are devoid of DYN immunoreactivity in this human model.

Species differences in the colocalization of KNDy peptides

We propose that the different colocalization patterns in the present human study and in previous animal experiments partly reflect species differences in reproductive mechanisms (28). These may include the absence of DYN signal from most KP neurons and fibers in humans, which is in contrast with the extensive coexpression in the rodent (3, 4, 14), sheep (5), and goat (8) ARC. Another putative species difference is the large excess of NKB-IR over KP-IR perikarya in the present human study, as opposed to the 2-fold excess in the ARC of male mice (4).

Sex-dependent variations in the absolute and relative abundances of KP and NKB and their colocalization pattern

In a previous study of KP-IR and NKB-IR neurons, we identified a series of sex-dependent morphological differences between aged human males and females (18). In particular, KP immunoreactivity was highly sexually dimorphic; the number of KP-IR perikarya, the density of KP-IR fibers, and the incidence of KP-IR afferents on GnRH neurons were much higher in aged women compared with men (18). It is worth noting that the NKB to KP neuron density ratio also differed, being 2.18 in aged males (>50 yr) and 1.55 in postmenopausal females (>55 yr) (18). In further support of the idea that the KP/NKB colocalization pattern is sexually dimorphic, the ratio of dual-labeled NKB-IR as well as KP-IR afferent contacts onto GnRH-IR neurons was significantly higher in postmenopausal women (∼25–30%) than in aged men (∼8–10%) (18). We proposed that sex differences are due to either organizational effects of sex steroids during critical period(s) of sexual differentiation or alternatively to the loss of negative sex steroid feedback in postmenopausal women, unlike in aged men who maintain relatively high testosterone levels. In our previous dual-immunofluorescent study, we demonstrated a high degree of overlap between the KP and NKB systems of postmenopausal women; more than 80% of KP-IR perikarya and NKB-IR perikarya contained also the other neuropeptide (17). The degree of this colocalization is likely to be sexually dimorphic and much lower in aged males whose Inf contained more than twice as many NKB-IR as KP-IR perikarya (18).

It is worth noting that the sexual dimorphism of KP and NKB neurons in the ARC/Inf region is not unique to humans. The ARC of the sheep contains higher NKB (29) and KP (1) cell numbers in females than in males. In rats, sex differences were reported in the projection fields of NKB-IR axons within the infundibular area (23).

Aging-dependent variations in the absolute and relative abundances of KP and NKB

The human NKB and KP systems, at least in the male, also exhibit robust aging-dependent changes. Our preliminary data indicate that the enhancements of the absolute NKB and KP cell numbers coincide with a significantly enhanced percentage of KP-expressing NKB neurons in aged men (Molnár et al., manuscript in preparation).

Role of NKB in the regulation of the human reproductive axis

The tachykinin peptide NKB plays a crucial role in human reproduction and inactivating mutations of the NKB and NK3 encoding genes cause normosmic hypogonadotropic hypogonadism (30, 31). Although the first reports did not indicate fertility deficits in the NK3 mutant mice (32), later analysis focusing on the reproductive phenotype noticed subfertility (33), suggesting functional similarities in NKB/NK3 signaling between the human and mouse species.

Out of the three KNDy peptides, NKB provided the heaviest immunohistochemical signal in the Inf and the InfS of young men (present report), aged men (18), and aged women (18). In immunoperoxidase-based studies, the dominance of NKB over KP was highest in young men (present study) in which the density of NKB-IR perikarya and fibers were about 5 times as high as those of the KP-IR elements and NKB-IR axons established about 6 times as many axosomatic and 5 times as many axodendritic contacts onto GnRH neurons as did KP-IR axons.

Conflicting results of previous experiments suggest that the net effect of NKB on LH secretion depends on animal species and endocrine paradigms. Intraperitoneal or intracerebroventricular NKB administration to male mice had no effect on serum LH (34), whereas the intracerebroventricular injection of the selective NK3 agonist senktide reduced LH secretion in ovariectomized rats treated with a low dose of estradiol (35). Reduced LH secretion in response to senktide was also observed in ovariectomized and in ovariectomized and estradiol treated rats (36) and in ovariectomized mice (3), whereas another study on rats found stimulatory effect on LH secretion in the presence of physiological levels of estradiol (37). Senktide also stimulated LH secretion in castrated male monkeys (16) and in the follicular, but not in the luteal, phase in ewes (38), whereas reduced net LH secretion was observed in ovariectomized goats (8).

Multiunit activity recorded in the ARC is considered to be an electrophysiological correlate of the GnRH pulse generator activity. The coordinated bursts of neuronal firing occur in synchrony with the LH secretory pulses in various animal species (8, 39), including primates (40). Senktide dose dependently suppressed the frequency of pulsatile LH secretion and inhibited hypothalamic multiunit activity volleys in ovariectomized rats, independently of gonadal steroid levels (36). In contrast, a robust increase in the frequency of multiunit activity volleys was observed in ovariectomized goats (8).

NKB mainly acts upstream from GnRH neurons. For example, the stimulatory effect of intravenous NKB and senktide on LH secretion could be abolished by the GnRH receptor antagonist acyline (16). One major target site for NKB actions appears to be on NKB (KNDy) neurons of the ARC/Inf. Accordingly, 1) we found numerous NKB-IR afferent contacts on human NKB neurons, 2) similar contacts were reported previously in rats (14) and sheep (5, 41), 3) NK3 receptors are present on these cells in rodents and sheep (3, 14, 42), and 4) KP (KNDy) neurons in the ARC of male mice respond with c-Fos expression (4) and depolarization (4) to senktide.

There is little agreement regarding the possibility that NKB also influences GnRH neurons directly. In sheep, GnRH neurons do not express NK3 immunoreactivity (42). In mice, although single-cell microarray and RT-PCR studies provided proof for NK3 mRNA expression in GnRH neurons (43), in situ hybridization studies were unable to confirm this finding (4). Also, senktide did not activate mouse KNDy neurons in vitro (4). In rats, although immunohistochemical studies found evidence for NK3 immunoreactivity in only 16% of GnRH-IR cell bodies (44), the receptor was more abundant on GnRH-IR axon terminals in the median eminence (44) where frequent appositions between GnRH-IR and NKB-IR axons occurred (23, 44). Although NKB in itself did not alter GnRH release from hypothalamic explants of male rats, it abrogated the KP-induced release of GnRH, suggesting a complex mode of action, which is likely parallel with, and not only upstream from, the KP action (45). Recent functional evidence from KISS1R-knockout mice indicates that intact KP/KISS1R signaling is required for the suppression of LH secretion by senktide. This finding provides support for the concept that the dominant action of NKB is upstream from KP neurons, instead of being exerted directly on GnRH cells (45).

Results of the present and a previous (18) human study indicate that NKB-IR axons abundantly innervate human GnRH neurons and the incidence of these contacts is several times as high as those of KP-IR axons. It will require clarification whether this anatomical pathway uses NKB/NK3 signaling. Alternatively, neurotransmitter(s) other than NKB may act in this communication, which is not likely to be KP or DYN, in view of their relative paucity in young male individuals. In addition to innervating GnRH-IR cell bodies and dendrites, NKB-IR axons also represented the most abundant KNDy peptide around the portal capillary plexuses of the human postinfundibular eminence (25). This hypothalamic site that lies outside the blood-brain barrier may represent an important site of interaction between NKB-IR and GnRH-IR axons. It also remains possible that NKB is released into the hypophysial portal circulation to influence adenohypophysial functions. In vitro evidence from rats, indeed, indicates that NKB can induce prolactin secretion from perifused pituitary cells (46).

Role of KP in the regulation of the human reproductive axis

In humans, KP/KISSR1 signaling plays a pivotal role in reproduction. Loss of function mutations of the genes encoding KISSR1 (47–49) and KP (50) result in hypogonadotropic hypogonadism. In recent models of the GnRH pulse generator, KP was proposed to provide the main output signal of the pulse generator neuronal network toward GnRH neurons, whereas NKB and DYN seem to primarily account for the intranuclear communication of KNDy neurons via acting on NK3 and KOR, respectively (3, 8). It is generally believed that independently from the species, KP acts directly on GnRH neurons that express KISS1R mRNA (10–12) and, in mice, respond with depolarization to KP (11, 51, 52).

As in laboratory and domestic animals, KP increases LH secretion in men (20, 53, 54) and women (55, 56), most potently during the preovulatory phase of the menstrual cycle in the latter. It is interesting to note that the continuous intravenous infusion of KP enhanced the LH pulse frequency in men (53), indicating that KP not only acts on GnRH neurons but also upstream from the pulse generator network. This finding suggests a species difference from the mouse in which KNDy neurons do not appear to synthesize KISS1R (57) and express only NK3 (3) and KOR (3) mRNAs.

In humans, axosomatic, axodendritic and axoaxonal contacts may serve as communication pathways between KP and GnRH neurons (17). In previous studies we showed that the KP system of aged human individuals exhibits a robust sexual dimorphism (18) with postmenopausal women having several times higher densities of KP-IR cell bodies and fibers in the Inf and higher incidences of KP-IR afferent contacts onto GnRH neurons than aged men above 50 yr. Preliminary data that KP immunoreactivity is even much lower in the hypothalamus of young men (Molnár et al., manuscript in preparation) led us to carry out the present study to challenge the validity of the KNDy neuron terminology and concept for the young male human model. Our present immunohistochemical data indicate that the density of KP-IR perikarya and fibers in the Inf were approximately 5 times lower than those of the corresponding NKB-IR elements and the number of KP-IR appositions to GnRH-IR cell bodies and dendrites only reached about one fifth and one sixth, respectively, of those established by NKB-IR axons. The functional consequences of the surprisingly low level of KP immunoreactivity in young men (in somata as well as fibers) require clarification. In view that KP is thought to represent the main neurotransmitter output of the pulse generator system (3, 8), its low level in the mediobasal hypothalamus of young men is compatible with only a moderate stimulation of GnRH/LH secretion. Much higher raw numbers of the KP-IR cell bodies, fibers, and contacts onto GnRH neurons in aged male human subjects (18) and their highest incidence in postmenopausal women (18) are in accordance with the idea that KP immunoreactivity and serum LH levels are linked. A future challenge will be to correlate the immunohistochemical images of KP and the other KNDy peptides with the GnRH neurosecretory output at the different human age and sex groups.

Absence of DYN immunoreactivity from most KP neurons and their fiber projections

The opioid peptide DYN is rather ubiquitous and may have multiple sites of action upon the reproductive axis. These sites are likely upstream from the GnRH neuron that does not appear to express KOR in rats (58). DYN is critically involved in progesterone negative feedback to GnRH neurons in ewes; the majority of DYN cells in the ARC of ovariectomized ewes contain progesterone receptor (59), and progesterone treatment increases preprodynorphin mRNA expression in the ARC and DYN levels in the cerebrospinal fluid (60).

DYN is an important regulator of the pulse generator system. In sheep, KOR antagonists stimulate the episodic secretion of LH during the luteal phase (61). In ovariectomized goats, central administration of DYN decreases and KOR antagonist increases the frequencies of the multiunit activity volleys and of the LH secretory pulses (8). Opioid peptides also regulate negatively the pulsatile release of prolactin and LH in humans; this inhibitory tone can be suspended by the blockade of opioid receptors with naloxone (62, 63).

The concept and terminology of the KNDy neurons rely on the similar results of colocalization experiments from several animal species. DYN has been detected in NKB (and/or KP) neurons in the ARC of sheep (5, 41), mice (3, 4), rats (14, 23), and goats (8). Moreover, the DYN receptor KOR is present in subsets of KNDy neurons in the ARC of mice (3, 4). Our present immunohistochemical study to address the presence of DYN immunoreactivity in human KP neurons was also informed by previous reports in which preprodynorphin mRNA expression was detected in the human Inf (64) and the monkey ARC (65).

The somewhat unexpected absence of DYN immunoreactivity in most KP-IR somata and fibers of young male humans questions the universal importance of DYN peptides within NKB and KP neurons of the Inf and reveals an important difference from the rodent, sheep, and goat species (1–5, 8, 23). It is worth noting that species also vary considerably regarding the sex steroid regulation of DYN in the ARC/Inf. Preprodynorphin expressing neurons showed reduced numbers in postmenopausal women (64) and in ovariectomized ewes (60). In contrast, there was no change in mRNA expression in postmenopausal monkeys (65) and preprodynorphin mRNA was increased in the absence of sex steroids in mice (3).

The absence of DYN immunoreactivity from most KP-IR neurons and their fibers we report in this study is unlikely to be entirely caused by the limited sensitivity of the applied immunohistochemical method because of the following: 1) DYN-IR cell bodies (e.g. magnocellular perikarya in the supraoptic nucleus) and fibers (e.g. a dense fiber plexus in the ventromedial nucleus) were readily detectable elsewhere in the hypothalamus, 2) substantial colocalization with KP was also undetectable using the highly sensitive tyramide signal amplification method to visualize DYN, or 3) using an antiserum against a different prodynorphin cleavage product, dynorphin B.

Summary of neuroanatomical findings

First, the regional density of NKB-IR cell bodies, fibers, and contacts onto GnRH neurons exceed about 5-fold those of KP-IR neuronal elements in the Inf.

Second, in addition to NKB-IR cell bodies and processes (in both the Inf and the InfS) that are devoid of KP labeling, KP-IR elements lacking NKB immunoreactivity are also highly abundant, as established in dual-immunofluorescent studies. In this study only 32.9 ± 4.7% of the NKB-IR and 75.2 ± 6.6% of the KP-IR perikarya were dual labeled.

Third, DYN-IR cell bodies and fibers occur much less frequently than either NKB-IR or KP-IR elements; KP-IR axons in the Inf and the InfS contain DYN immunoreactivity only occasionally.

In conclusion, the immunohistochemical observations we made on hypothalamic tissue samples of young male human subjects question the universal validity of the KNDy neuron concept and terminology and suggest that the abundance of these peptides and their overlap are species, sex, and age dependent.

Acknowledgments

We thank Mr. László Barna (the Nikon Microscopy Center at Institute of Experimental Medicine), Nikon Austria GmbH, and Auro-Science Consulting Ltd. for kindly providing microscopy support. We also thank Ms. Hajni Bekó for expert technical assistance.

The research leading to these results has received funding from the National Science Foundation of Hungary (Grants OTKA K83710, K100722) and the European Community's Seventh Framework Program (FP7/2007–2013) under Grant 245009. W.S.D. is supported by a National Institute of Health Research Career Development Fellowship.

Disclosure Summary: All of the authors have nothing to disclose.

Footnotes

Abbreviations:

ARC: Arcuate nucleus
DYN: dynorphin A
FITC: fluorescein isothiocyanate
Inf: infundibular nucleus
InfS: infundibular stalk
IR: immunoreactive
KISS1R: KP receptor
KNDy: KP/NKB/DYN A
KOR: DYN receptor
KP: kisspeptin
NK3: NKB receptor
NKB: neurokinin B.

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PERMALINK

Low Degree of Overlap Between Kisspeptin, Neurokinin B, and Dynorphin Immunoreactivities in the Infundibular Nucleus of Young Male Human Subjects Challenges the KNDy Neuron Concept

Erik Hrabovszky

Máté T Sipos

Csilla S Molnár

Philippe Ciofi

Beáta Á Borsay

Péter Gergely

László Herczeg

Stephen R Bloom

Mohammad A Ghatei

Waljit S Dhillo

Zsolt Liposits

Abstract

Materials and Methods

Human subjects

Section preparation

Pretreatments

Immunohistochemical detection of KP

Immunohistochemical detection of NKB and DYN

Dual-immunoperoxidase detection of KP and GnRH or NKB and GnRH

Fluorescent immunohistochemistry

Section mounting and coverslipping

Digital photography

Experiment 1. Studies of the incidences and overlaps of IR cell bodies in the Inf

Experiment 2. Studies of the regional abundance of IR fibers in the Inf

Experiment 3. Studies of immunoreactive fibers in the InfS

Experiment 4. Studies of the incidences of KP-IR and NKB-IR appositions onto GnRH-IR neurons of the Inf

Results

Fig. 1.

Fig. 2.

Experiment 1. Incidence of IR perikarya in the Inf

Fig. 5.

Fig. 3.

Experiment 2. Abundance of IR fibers in the Inf

Fig. 6.

Experiment 3. Abundance of immunoreactive fibers in the InfS

Experiment 4. Frequency of KP-IR and NKB-IR appositions onto GnRH-IR neurons

Fig. 4.

Fig. 7.

Discussion

Species differences in the colocalization of KNDy peptides

Sex-dependent variations in the absolute and relative abundances of KP and NKB and their colocalization pattern

Aging-dependent variations in the absolute and relative abundances of KP and NKB

Role of NKB in the regulation of the human reproductive axis

Role of KP in the regulation of the human reproductive axis

Absence of DYN immunoreactivity from most KP neurons and their fiber projections

Summary of neuroanatomical findings

Acknowledgments

Footnotes

References

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