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Centre for Neuroendocrinology, Department of Physiology, School of Medical Sciences, University of Otago, Dunedin 9054, New Zealand
Address all correspondence and requests for reprints to: Allan E. Herbison, Centre for Neuroendocrinology, Department of Physiology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin 9054, New Zealand. E-mail: allan.herbison{at}stonebow.otago.ac.nz.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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If kisspeptin neurons play a central role in pubertal activation, then it will be critical to obtain a detailed understanding of kisspeptin neurons and how they interact with GnRH neurons. In situ hybridization and immunocytochemical studies in mice, rats, sheep, and primates have shown that cells expressing Kiss1 mRNA are clustered in three regions of the hypothalamus: the periventricular preoptic area, the dorsomedial hypothalamus, and the arcuate nucleus (ARN) (9, 17, 18, 19, 20, 21, 22). Importantly, whole hypothalamic levels of both kisspeptin and GPR54 mRNA are known to increase across postnatal development (9, 23), suggesting that kisspeptin-GPR54 signaling in the hypothalamus is up-regulated around the time of puberty.
Intriguingly, GPR54 transcript levels in GnRH neurons do not appear to change across puberty (16). In contrast, the number of kisspeptin mRNA-expressing cells increases 7-fold in the anteroventral periventricular nucleus (AVPV) over this period (16). Because the AVPV is a brain region that contains neurons projecting directly to GnRH neurons (24, 25), it seems reasonable to suggest that an increase in the number of AVPV kisspeptin neurons projecting to GnRH neurons may contribute to GnRH neuron activation around the time of puberty (16).
In the present study, we tested the hypothesis that GnRH neurons receive increased numbers of kisspeptin inputs over postnatal development. To achieve this, we needed to develop an immunocytochemical paradigm that enabled us to examine the relationship between kisspeptin fibers and GnRH somata. Few kisspeptin antisera are available, and, until recently, these have all lacked sensitivity for immunocytochemical work in rodent brains. For example, a substantial population of kisspeptin mRNA-expressing cells exists in the AVPV and periventricular regions of the rodent hypothalamus (21, 26), but no antibody published to date has been able to detect kisspeptin immunoreactivity in these areas (17, 18). Using a new antisera directed against the murine kisspeptin-10 (19), we report here the distribution of kisspeptin cell bodies and fibers within the hypothalamus of the male and female mouse across postnatal development.
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Materials and Methods |
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Immunocytochemistry
Animals were anesthetized with sodium pentobarbital (3 mg/100 µl, ip) and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.6). The brains were removed and postfixed in the same fixative for 60 min, then transferred to a 30% sucrose/Tris-buffered saline (TBS; 0.2 M Tris, 0.15 M sodium chloride) solution overnight. The following day, brains were frozen on the stage of a sliding microtome, and three sets of coronal sections 30-µm thick were cut from the level of the medial septum (MS) through to the end of the hypothalamus. Mice of different ages and sex were processed simultaneously.
Single label immunocytochemistry
Two sets of sections were treated with 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity, and then washed three times in TBS (10 min/wash). Sections were then incubated for 48 h at 4 C in a polyclonal rabbit anti-kisspeptin-10 antiserum (1:5,000; no. 566, gift from A. Caraty, Tours, France) in TBS containing 0.3% Triton X-100, 0.25% BSA, and 2% normal goat serum. Sections were then washed three times in TBS (10 min/wash) before being incubated in a biotinylated goat antirabbit secondary antibody (Vector Laboratories, Inc., Burlingame, CA) at 1:400 in TBS containing 0.3% Triton X-100 and 0.25% BSA for 90 min at room temperature. After subsequent washing in TBS, the sections were incubated in Vector Elite avidin-peroxidase (Vector) at 1:100 in TBS containing 0.3% Triton X-100 and 0.25% BSA for 90 min at room temperature. The sections were again washed and immunoreactivity was revealed using glucose-oxidase, nickel-enhanced diaminobenzidine hydrochloride. The sections were washed thoroughly in TBS, mounted onto gelatin-coated glass slides, air dried, dehydrated in ethanol followed by xylene, and then coverslipped with DPX.
Dual-label immunofluorescence
The remaining set of sections was washed thoroughly in TBS and incubated for 48 h at 4 C in the polyclonal rabbit anti-kisspeptin-10 antiserum (1:2000) in TBS containing 0.3% Triton X-100, 0.25% BSA, and 2% normal goat serum. Sections were then washed three times in TBS (10 min/wash) before being incubated in a biotinylated goat antirabbit secondary antibody (Vector) at 1:400 in TBS containing 0.3% Triton X-100 and 0.25% BSA for 90 min at room temperature. After subsequent washing, the sections were incubated in a streptavidin-conjugated 568 (Alexa Fluor, Molecular Probes, Eugene, OR) at 1:200 in TBS containing 0.3% Triton X-100 and 0.25% BSA for 90 min at room temperature. The tissue was then washed in TBS and incubated for 48 h at 4 C in a polyclonal chicken anti-GFP antiserum (Chemicon International, Inc., Temecula, CA) at 1:2500 in TBS containing 0.3% Triton X-100, 0.25% BSA, and 2% normal goat serum. Sections were then washed three times in TBS before being incubated in a goat antichicken conjugated 488 (Alexa Fluor, Molecular Probes) at 1:200 in TBS containing 0.3% Triton X-100 and 0.25% BSA for 90 min at room temperature. The sections were washed thoroughly in TBS, mounted onto gelatin-coated glass slides, air dried, and coverslipped with Vectashield aqueous mountant, and the coverslip was sealed with nail polish.
Controls and specificity
The production and characterization of the kisspeptin-10 antibody has been published (19). In brief, mouse kisspeptin-10 (YNWNSFGLRY-NH2) was coupled to BSA using glutaraldehyde and used as an immunogen in rabbits. The antiserum is highly specific to mouse kisspeptin-10 with RIA binding not inhibited by any one of eight different hypothalamic peptides including other RFamides such as prolactin-releasing peptide. Similarly, immunoreactivity is abolished by preadsorption of the antiserum with 1 µM kisspeptin-10 but not 1–10 µM prolactin-releasing peptide. Controls for this series of experiments included omission of the primary antibody in single and dual-label experiments and use of the kisspeptin-10 antibody preadsorbed overnight with 1 µM murine kisspeptin-10 peptide (a gift from A. Caraty).
Analysis
Sections were examined with an Olympus BX51 microscope using either bright-field or epifluorescence microscopy. Analysis of the single-labeled tissue was undertaken by counting the number of kisspeptin-immunoreactive cell bodies located within the AVPV and the preoptic periventricular nucleus (PeN) divided into rostral and caudal regions (rPeN and cPeN, respectively; Fig. 1
). As assessed from our confocal microscopy studies (see below), kisspeptin neurons exhibit a mean (±SEM) diameter of 13.8 ± 0.5 µm (n = 17). As such, two coronal brain sections, 30 µm apart, at each level were analyzed in each mouse to avoid any double counting errors. In each section, the total number of immunoreactive cells exhibiting cytoplasmic staining with a region of nuclear exclusion or, in the case of heavily labeled cells, immunoreactive cells with a round or oval cytoplasmic profile, were counted. The anteroposterior levels for each region are represented by Figs. 28–29, 30, and 31–32, respectively, of Paxinos and Franklin (Ref. 29). The same procedure was undertaken for the dorsomedial nucleus (DMN) analysis, where all immunoreactive cells in the dorsomedial hypothalami were counted at the level of Figs. 43 and 44 in Ref. 29) (see Fig. 1E). Mean cell counts for each mouse were determined and grouped to provide mean (±SEM) values. Statistical analysis was undertaken using ANOVA with post hoc Student-Newman-Keuls tests.
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). Using a 40x objective, each GnRH neuron was then evaluated for close apposition to a kisspeptin fiber. To be considered in close apposition, the kisspeptin fiber was required to be directly adjacent to the GnRH neuron cell body and/or proximal dendrite in the same plane of focus. Two sections from each mouse at each of these three levels, 60 µm apart, were counted (Figs. 21–23, 24–25, and 26–28, respectively, of Paxinos and Franklin; Ref. 29), and the percentage of GnRH neurons showing close appositions with kisspeptin fibers was determined. Statistical analysis was undertaken using ANOVA with post hoc Student-Newman-Keuls tests.
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Results |
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Three populations of kisspeptin-10-immunoreactive cell bodies were identified in the coronal sections. The first and largest population comprised a continuum of kisspeptin cell bodies lying close to the third ventricle, extending from the AVPV into the preoptic PeN (Fig. 1
, A–C). A second, smaller population was observed scattered within the dorsomedial hypothalamus and anterior hypothalamus, referred to here as the DMN group (Fig. 1E). The third small population of cell bodies was located in the ventrolateral ARN (Fig. 2C).
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, E and F, and 2C). Whereas modest staining was detected within the internal zone of the median eminence, it is noteworthy that none was found in the external zone of the median eminence (Fig. 2C). Elsewhere in the hypothalamus, kisspeptin fiber staining was notable within the AVPV and PeN (Fig. 2G) and extending dorsally and laterally from these nuclei into adjacent brain regions (Figs. 1, A–C, and 2A), as well as within the DMN. There was a conspicuous absence of fiber staining in the ventromedial nucleus (Fig. 1F). Within the preoptic area, fibers were found in the median preoptic nucleus (Fig. 2A), the medial preoptic area, and coursed through the DBB to the ventral-lateral septum and the anterior portion of the bed nucleus of the stria terminalis. No fibers were detected in the MS. Scattered fibers were also found in the subfornical organ and paraventricular thalamic nucleus, as well as within the supraoptic (Fig. 2E) and paraventricular (Fig. 2F) nuclei.
Tissue that underwent immunocytochemistry with either the omission of the primary antibody or incubation with primary antibody that had been preadsorbed with the kisspeptin peptide resulted in complete absence of labeling (Figs. 1D and 2, B and D).
A marked sexual dimorphism in kisspeptin immunoreactivity exists in the AVPV/PeN of the adult mouse
Sex differences in kisspeptin staining were observed in the rostral hypothalamus of adult mice (n = 5 male, n = 4 female; Fig. 3, A and B). Whereas the overall distribution of kisspeptin-10 immunoreactivity was very similar between males and females, the number of cell bodies detected in the AVPV, rPeN, and cPeN was highly sexually dimorphic with over 10-fold more cell bodies detected in the female (P < 0.001; Fig. 3). The density of kisspeptin fibers in the lateral septum, DBB, bed nucleus of the stria terminalis, preoptic and anterior hypothalamic areas (Fig. 3, A and B) was lower in males compared with females. In contrast, the pattern and density of kisspeptin staining in the ARN was similar in males and females, and no sex differences were found in the numbers of cell bodies located in the DMN (males, 14.9 ± 2.6 cells per section; females, 11.8 ± 1.0 cells per section).
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Male and female mice showed a similar pattern of postnatal development of kisspeptin-immunoreactive cell numbers in the AVPV, rPeN, and cPeN (Figs. 4 and 5). In male mice, no kisspeptin-immunoreactive cells were detected in the AVPV/PeN at P10, with only small numbers detected at P25. However, between P25 and P31, there was an approximately 500% increase in kisspeptin cell numbers throughout the AVPV/PeN (P < 0.01; Fig. 5A). This trend continued as a smaller 30–50% increase between P31 and P45 (P < 0.01) with peripubertal P45 mice not being different from adults (Fig. 5A). In females, essentially no kisspeptin neurons were found in the AVPV/PeN at P10, but cells were clearly evident at P25 (P < 0.01; Fig. 4A). There was then a doubling of kisspeptin cell numbers between P25 and P31 (P < 0.01) to adult levels in the PeN (Figs. 4, B and C, and 5B). In the AVPV, however, kisspeptin cell numbers did not achieve adult-like levels until after the onset of puberty, with P61 numbers being approximately double those of P31 female mice (P < 0.001; Fig. 5B). Although not quantified, an increase in the density of kisspeptin fibers accompanying the kisspeptin cell bodies in periventricular regions was evident (Fig. 2, G and H).
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The numbers of kisspeptin-immunoreactive cells in the DMN of males and females exhibited decreasing numbers of cells with postnatal development (P10 = 23.6 ± 2.9; P25 = 12.4 ± 1.2; P61 = 14.9 ± 2.6; P10 vs. P25 and P61; P < 0.05).
Development of kisspeptin inputs to GnRH neurons
Dual-labeling for kisspeptin-10 and GnRH was undertaken using a chicken GFP antibody to detect GFP in the Spergel GnRH-GFP transgenic mouse line. In our hands, GFP staining identifies 100% of GnRH neurons, with "ectopic" GFP-expressing cells clearly localized to the lateral septum in this mouse line. As mentioned, the advantage of using this approach is that more of the GnRH neuron dendrite can be visualized. Dual-labeling revealed close appositions between kisspeptin-10-immunoreactive fibers and GnRH neuron somata and dendrites (Fig. 6A). The omission of the kisspeptin antisera resulted in a complete absence of red immunofluorescence. Eight randomly selected GnRH neurons, defined previously to exhibit close appositions with epifluorescence microscopy, were evaluated further using confocal microscopy. Each of these cells was confirmed to exhibit close appositions at the level of the confocal (Fig. 6A).
In adult female mice, the GnRH neurons with kisspeptin appositions were located in specific brain regions (Fig. 6B). In the rPOA, 40 ± 7% of GnRH cell bodies were detected to have kisspeptin fiber appositions (Figs. 5, A and B, and 6, A and B). In contrast, only 12 ± 5% and 10 ± 4% of GnRH neurons in the hDBB and vDBB, respectively, had appositions, and no GnRH neurons located in the MS were detected to be in close apposition to a kisspeptin fiber. A similar overall topography existed in adult male mice with 10 ± 3% of GnRH neurons in the rPOA having kisspeptin appositions, whereas none of the more rostral GnRH neurons in the MS and DBB had appositions. The overall percentage of rPOA GnRH neurons with kisspeptin appositions was significantly less in males compared with females (P < 0.05). The numbers of GnRH neurons detected in the MS, DBB, and rPOA were not different between adult males and females (rPOA; 15.2 ± 2.6 GnRH neurons per section in males compared with 18.0 ± 2.8 in females).
In terms of postnatal development, there was complete absence of kisspeptin fibers within the MS/DBB/rPOA of male and female P10 mice, and only a very few rPOA GnRH neurons exhibited kisspeptin appositions at P25 (Fig. 6C). From P25 onward, however, there was a significant (P < 0.001) marked increase in the number of rPOA GnRH neurons with kisspeptin contacts between P25 (2.3 ± 0.9% of GnRH neurons) and P31 (24 ± 3%) and a further significant 80% increment to P61 (40 ± 7%; P < 0.001; Fig. 6C) in female mice. A similar trend was observed in male mice, where rPOA GnRH neurons in adults had significantly more kisspeptin appositions compared with P31 or younger mice (P < 0.001; Fig. 6C). GnRH neurons located in the MS and DBB did not exhibit kisspeptin appositions before puberty in either sex.
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Discussion |
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The distribution of kisspeptin-10-immunoreactive cells in the rostral hypothalamus reported here with this new antibody is in excellent agreement with Kiss1 mRNA in situ hybridization studies in the mouse. In those studies, a large population of Kiss1 mRNA-expressing cells was detected within the AVPV and PeN of adult male and female mice (21, 22, 26). It remains unclear why other kisspeptin antibodies have failed to detect this substantial population of kisspeptin neurons (17) but the use here of an antibody generated against mouse kisspeptin-10 in mouse tissue may be significant. In situ hybridization analyses also detected a population of Kiss1 mRNA-expressing cells in the ARN (22, 26). Abundant kisspeptin immunoreactivity was also found in the ARN, although relatively few clearly labeled cell bodies were observed. This is likely due to the very high density of kisspeptin fibers in the ARN that made it difficult to discern individual cell bodies, and the observation that kisspeptin biosynthesis is robustly suppressed by gonadal steroids in the ARN of intact male and female mice (22, 26). In our preliminary experiments, we have examined kisspeptin immunoreactivity in ovariectomized mice and observed a large population of kisspeptin-immunoreactive cell bodies in the ARN. We also noted a third population of kisspeptin-immunoreactive cells scattered within the dorsomedial hypothalamus, as has been seen in the sheep (19, 20) and rat (17). Interestingly, these cells have not been reported on in Kiss1 mRNA in situ hybridization experiments as yet (21, 22, 26).
Previous studies have shown that Kiss1 mRNA levels within the whole hypothalamus of the rat and primate fluctuate over the course of postnatal development (9, 23). We show here a clear developmental increase in kisspeptin expression within the AVPV/PeN continuum. Our earlier investigation found that the numbers of Kiss1 mRNA-expressing cells located in the AVPV increased 7-fold between P18 and adulthood in male mice (16). We now extend this result to show that: 1) the same pattern of development (5-fold increase from P25 to adulthood in males) occurs for kisspeptin peptide-containing cells in the AVPV; 2) this also occurs in the AVPV of the female mouse; and 3) this developmental profile is also exhibited by the much larger population of kisspeptin neurons found within the PeN. Kisspeptin-immunoreactive cell numbers in the DMN exhibited a completely different developmental pattern, and fiber staining was evident in the ARN at all postnatal ages. Prior data indicate that the numbers of Kiss1 mRNA-expressing cells in the ARN region did not change between P18 and adulthood in male mice (16). These observations demonstrate that it is only the kisspeptin neurons of the AVPV/PeN that exhibit a postnatal developmental increase in kisspeptin synthesis.
Details of the ontogeny of kisspeptin signaling at a cellular level are only just emerging. Our earlier study indicated that more than 90% of prepubertal and adult male GnRH neurons expressed GPR54 mRNA, but that the percentage of GnRH neurons responding electrophysiologically to kisspeptin increased from 27% in prepubertal mice to nearly 100% in adult males (16). Alongside evidence for a substantial increase in the numbers of Kiss1 mRNA-expressing cells in the AVPV (16), we suggested that a two-step mechanism for kisspeptin activation of GnRH neurons may exist, involving 1) a developmental change in the coupling of GPR54 to its effector pathways within GnRH neurons, and 2) the development of kisspeptin inputs to GnRH neurons. Our present results provide the first direct evidence in support of the latter part of the mechanism. Appositions between kisspeptin fibers and GnRH neuron somata are essentially absent before P25 but then appear over the next few days leading up to puberty. It is important to note that the percentages of GnRH neurons estimated to receive direct kisspeptin inputs in this study are not absolute and are likely to be underestimates. On one hand, we have not been able to evaluate kisspeptin inputs to the distal dendritic tree of GnRH neurons (31), whereas on the other, dual immunofluorescence is unlikely to be sensitive enough to detect every kisspeptin-containing fiber innervating a GnRH neuron. Nevertheless, the present observations provide relative estimates of kisspeptin inputs to GnRH somata and proximal dendrites on the basis of topography, development, and sex. Given the potent effects of kisspeptin on the excitability of GnRH neurons at all postnatal ages (16), the peri-pubertal timing of the development of kisspeptin inputs to rPOA GnRH neurons would be well positioned to activate GnRH neurons to achieve puberty onset.
The strong correlation between the emergence of kisspeptin-synthesizing neurons in the AVPV/PeN and the appearance of kisspeptin fibers adjacent to GnRH neurons over postnatal development strongly suggests that it is the AVPV/PeN neurons that provide the kisspeptin inputs to GnRH neurons. Interestingly, these kisspeptin inputs appear to target GnRH neurons located in the rPOA and not those located in the MS. Anterograde labeling studies have demonstrated that AVPV neurons project to rPOA GnRH neurons from as early as embryonic day 18 in the rat (24). Thus, the apparent "abrupt" innervation of rPOA GnRH neurons by kisspeptin fibers from P25 onward may result from either existing AVPV/PeN afferents to GnRH neurons turning on kisspeptin synthesis at this time or the de novo innervation of GnRH neurons by kisspeptin fibers.
It is important to note that kisspeptin fibers are distributed throughout multiple hypothalamic nuclei and that kisspeptin inputs to GnRH neuron cell bodies represent only a small minority of these fibers. Alongside the similarly widespread distribution of GPR54 mRNA in the hypothalamus (9), this observation indicates that kisspeptin-GPR54 signaling is very likely to be involved in the regulation of multiple hypothalamic networks. It is of interest, therefore, that a developmental increase in kisspeptin fiber density occurs in several regions of the hypothalamus, suggesting that the peripubertal development of the AVPV/PeN kisspeptin network may have a wide impact upon hypothalamic functioning.
It remains to be established what underlies the development of the AVPV/PeN kisspeptin neuronal network. Although kisspeptin neurons in this region are positively regulated by gonadal steroids in adult mice (22, 26), it is unlikely that the peripubertal increase in gonadal steroid levels is responsible for the developmental increase in AVPV/PeN kisspeptin expression. The increments in periventricular kisspeptin cell number begin up to 2 wk before puberty onset when circulating estrogen or androgen levels remain low. Nevertheless, it will be interesting to assess the postnatal development of kisspeptin neurons in neonatally gonadectomized mice.
We report here a marked sexual dimorphism in kisspeptin expression exclusively within the AVPV/PeN of the hypothalamus. Previous in situ hybridization experiments undertaken separately in male and female mice had suggested the presence of a sex difference in Kiss1 mRNA expression in this area (22, 26). Overall, adult female mice exhibited 10-fold greater numbers of kisspeptin neurons in the AVPV/PeN compared with males. This pattern of sexual dimorphism is not unusual in the AVPV, where several different neurochemically defined neuronal populations exhibit sex differences that are larger in the female (32). However, to our knowledge, this is the first description of such a sex difference in the PeN. Indeed, the sexually dimorphic kisspeptin neuronal population appears to exist as a continuum within the AVPV and PeN. The functional significance of this sexual dimorphism is not known, but the recent suggestion that kisspeptin may be involved in generating the preovulatory LH surge in adult females (18, 33) may be relevant. Female rodents are thought to possess a sexually differentiated, estrogen-receptive neuronal population located in the AVPV that is responsible for generating the GnRH surge (32, 34). The kisspeptin neurons located in the AVPV (26)/PeN (Clarkson, J., and A. E. Herbison, unpublished observations) continuum of the female express estrogen receptor 
, the critical estrogen receptor isoform required for estrogen-positive feedback (35), exhibit increased levels of Kiss-1 mRNA before ovulation (33), and are suggested here to project directly to rPOA GnRH neurons in a sexually dimorphic manner. Although the brain regions critical for puberty onset are not known, it is possible that the kisspeptin neurons of the AVPV, and possibly the PeN, are involved in the activation of the GnRH neurons both at puberty and, later, during each cycle to generate the preovulatory GnRH surge.
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Acknowledgments |
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Footnotes |
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Author Disclosure Statement: The authors have nothing to declare.
First Published Online September 7, 2006
Abbreviations: ARN, Arcuate nucleus; AVPV, anteroventral periventricular nucleus; c, caudal; DBB, diagonal band of Broca; DMN, dorsomedial nucleus; hDBB, horizontal DBB; MS, medial septum; P, postnatal day; PeN, periventricular nucleus; r, rostral; rPOA, rostral preoptic area; vDBB, vertical DBB.
Received June 12, 2006.
Accepted for publication August 30, 2006.
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R. Lapatto, J. C. Pallais, D. Zhang, Y.-M. Chan, A. Mahan, F. Cerrato, W. W. Le, G. E. Hoffman, and S. B. Seminara Kiss1 / Mice Exhibit More Variable Hypogonadism than Gpr54 / Mice Endocrinology, October 1, 2007; 148(10): 4927 - 4936. [Abstract] [Full Text] [PDF]
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R. M. Luque, R. D. Kineman, and M. Tena-Sempere Regulation of Hypothalamic Expression of KiSS-1 and GPR54 Genes by Metabolic Factors: Analyses Using Mouse Models and a Cell Line Endocrinology, October 1, 2007; 148(10): 4601 - 4611. [Abstract] [Full Text] [PDF]
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