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Immunoelectron microscopic observation of the subcellular localization of kisspeptin, neurokinin B and dynorphin A in KNDy neurons in the arcuate nucleus of the female rat
Neuroscience Letters 612 (2016) 161–166
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Research paper
Immunoelectron microscopic observation of the subcellular localization of kisspeptin, neurokinin B and dynorphin A in KNDy neurons in the arcuate nucleus of the female rat Hiroko Murakawa a,b , Kinuyo Iwata a , Toshiyuki Takeshita b , Hitoshi Ozawa a,∗ a
Department of Anatomy and Neurology, Graduate School of Medicine, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, Japan Department of Reproductive Medicine, Perinatology and Gynecologic Oncology, Graduate School of Medicine, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, Japan b
h i g h l i g h t s • ARC kisspeptin neurons co-express with NKB and DynA. • Subcellular localization of kisspeptin, NKB and DynA in KNDy neurons were observed by immunoelectron microscopy. • Kisspeptin, NKB and DynA are contained within separate individual neurosecretory vesicles.
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Article history: Received 23 October 2015 Received in revised form 25 November 2015 Accepted 4 December 2015 Available online 8 December 2015 Keywords: GnRH/LH pulse KNDy neuron Immunoelectron microscopy Neurosecretory vesicle Reproduction
a b s t r a c t KNDy neurons are named for their co-expression of three neuropeptides, kisspeptin, neurokinin B (NKB) and dynorphin A (DynA). These cells, located in the hypothalamic arcuate nucleus (ARC), are associated with generation of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulses to control follicular growth and steroidogenesis. However, subcellular sorting mechanisms for secretory vesicles containing these neuropeptides have not been elucidated. In this study, we analyzed the localization pattern of kisspeptin, NKB and DynA in the ARC of the ovariectomized rat immediately treated with estrogen using immunoelectron microscopy. First, we identified neuropeptides by dual-labeled fluorescence immunohistochemistry, with results indicating all three neuropeptides co-express within individual ARC cells in female rats. Next, we investigated the subcellular localization pattern of kisspeptin, NKB, and/or DynA using post-embedding double immunoelectron microscopy, indicating that each type of neuropeptide is contained within separate and individual neurosecretory vesicles. This suggests sorting and packaging of kisspeptin, NKB and DynA is differentially regulated within KNDy neurons. Our findings facilitate understanding of regulatory mechanisms underlying kisspeptin secretion in KNDy neurons, and generation of GnRH/LH pulses induced by kisspeptin in the ARC. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulses are necessary for reproduction [1]. Abnormal LH pulses have been observed in women with polycystic ovary syndrome (PCOS) [2] and weight loss-related amenorrhea [3]. Thus, GnRH/LH
∗ Corresponding author at: Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 1138602, Japan. Fax: +81 3 5685 6640. E-mail address: [email protected] (H. Ozawa). http://dx.doi.org/10.1016/j.neulet.2015.12.008 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
pulse abnormalities have the potential to cause menstrual disorders, anovulation and infertility. Recent studies suggest pulsatile GnRH/LH secretion is generated by KNDy neurons [4]. These cells, which co-express kisspeptin, neurokinin B (NKB) and dynorphin A (DynA), have been identified in the hypothalamic arcuate nucleus (ARC) of mice [5], rats [6], ewes [7] and goats [8]. Kisspeptin fibers project, in a non-synaptic manner, to GnRH terminals located within the median eminence (ME) of monkeys [9], rats and goats [10,11]. GnRH neurons expressing kisspeptin receptors (Kiss1r) have been identified in mice [12] and rats [13]. KNDy neurons also express estrogen receptor ␣ (ER␣)
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in mice [14], rats [6,15] and ewes [16], and receive to negative feedback of estrogen to regulate LH release [14,15]. NKB also induces LH release, as evidenced by the ability of a selective NKB receptor (NK3R) agonist, senktide, to increase LH secretion in sheep [17] and multiple-unit electrical activity (MUA) volleys in the ARC of goats [8]. In contrast, DynA inhibits LH release, as demonstrated by the capacity of a selective DynA receptor (KOR) antagonist, nor-binaltorphimine (nor-BNI), to increase LH pulse frequency in sheep [18]. Further, MUA volleys in the ARC are inhibited by DynA and increased by nor-BNI in goats [8]. As KNDy fibers have been observed in close apposition to KNDy cell bodies in the ARC of rats [6], ovine [19] and goats [8], it is likely KNDy neurons communicate with each other. KNDy neurons express NK3R in mice [5], rats [6,20], and sheep [21]; KOR in mice [5]; but, do not express Kiss1r in mice [22] and ovine [23]. In rats, GnRH neurons do not express KOR and only slightly express NK3R [20,24]; while in sheep, these cells do not express NK3R [21]. Further, as senktide was shown to have no effect on GnRH neuronal firing in castrated male mice [25], GnRH neurons appear to be unaffected by NKB or DynA. Thus, KNDy neurons are thought to act as GnRH/LH pulse generators within neural circuits by regulating kisspeptin secretion through stimulation by NKB/NK3R signaling and inhibition by DynA/KOR signaling. Though co-expression of kisspeptin, NKB and DynA has previously been reported in mice [5], ewes [7] and goats [8], studies examining rat KNDy neurons indicate only proNKB and prodynorphin co-localize within ARC kisspeptin neurons [6]. Moreover, subcellular localization of neuropeptides within these cells has not been elucidated. Thus, in the current study, we investigated the subcellular localization of kisspeptin, NKB and DynA within KNDy neurons of female rats by double immunoelectron microscopy. 2. Materials and methods 2.1. Animals Adult Wistar-Imamichi strain female rats, weighing 200–250 g, aged 9–14 weeks (Institute for Animal Reproduction, Ibaraki, Japan), were housed in an air-conditioned room (23 ± 1 ◦ C) under 14 h light (6:00–20:00)/10 h dark (20:00–6:00) cycles with access to food and water. Timing of estrous cycle was monitored by daily vaginal smear cytology. Animals demonstrating at least two consecutive, regular 4-day estrous cycles were used in subsequent experiments. Animals were ovariectomized (OVX) and immediately received a Silastic tube implant filled with 20 g/ml -estradiol (E2; Sigma–Aldrich, St. Louis, MO) dissolved in sesame oil, for 7–13 days. As kisspeptin-immunoreactive (ir) and NKB-ir areas in the ARC are more prevalent in OVX + low E2 rats compared with OVX rats [26,27], and preliminary studies indicated DynA-ir areas in OVX + E2 rats were also larger than that of OVX rats, E2 implants were intended to yield plasma E2 levels similar to diestrus [28]. All surgeries were conducted under inhalational anesthesia using isoflurane. For perfusion fixation, animals were deeply anesthetized with sodium pentobarbital (50 mg/kg). Experimental procedures were conducted in accordance with Nippon Medical School’s Guidance on Animal Bioethics (based on guidelines for humane treatment of experimental animals issued by the US National Institutes of Health) and with approval from Nippon Medical School’s Committee on the Care and Use of Experimental Animals. 2.2. Antibodies Primary antibodies included mouse monoclonal anti-kisspeptin antibody (kindly provided by Dr. Ohtaki, Takeda Pharmaceutical
Co., Osaka, Japan) [29], rabbit polyclonal anti-NKB antibody (Novus Biological, Littleton, CO) [30], and rabbit anti-dynorphin A antibody (Phoenix Pharmaceuticals, Inc. Burlingame, CA) [31]. For fluorescent immunohistochemistry, secondary antibodies included Alexa Fluor 568-conjugated donkey anti-mouse immunoglobulin (IgG) (dilution 1:500; Molecular Probes, Eugene, OR), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:500; Molecular Probes), and Alexa Fluor 568-conjugated antirabbit IgG (Zenon kit, Molecular Probes). For post-embedding immunoelectron microscopy, secondary antibodies included goat anti-rabbit IgG labeled with 5 or 15 nm colloidal gold (BB International, Cardiff, UK), goat F(ab)2 anti-rabbit IgG labeled with 10 nm colloidal gold (BB International) and goat anti-mouse IgG (H + L) labeled with 10 or 15 nm colloidal gold (BB International), used at a dilution of 1:50. Dual-labeled fluorescence immunohistochemistry for kisspeptin and NKB, kisspeptin and DynA, or NKB and DynA in the ARC. Animals (n = 2) were perfused through the heart with physiological saline, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS) (pH 7.4). Brains were removed, post-fixed for 24 h in the same fixative at 4 ◦ C, then cryoprotected in 0.1 M PBS containing 30% sucrose and 0.9% NaCl at 4 ◦ C. Using a cryostat, brains were cut into 40-m-thick coronal sections (CM3050S, Leica Microsystems, Wezlar, Germany) before storage in cryoprotectant solution at −20 ◦ C until use in immunofluorescence studies. To perform double labeling for kisspeptin and either NKB or DynA, sections were incubated with primary mouse anti-kisspeptin antibody (1:5000) and either rabbit anti-NKB antibody (1:5000) or rabbit anti-DynA antibody (1:5000) for 4 days at 4 ◦ C. After they had been washed with PBS, sections were incubated with Alexa Fluor 568-conjugated donkey anti-mouse IgG and Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibodies for 2 h at room temperature (RT). To perform double labeling for DynA and NKB, sections were incubated with rabbit anti-DynA antibody (1:5000) at 4 ◦ C for 4 days. Sections were rinsed with PBS and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1:500) for 2 h at RT. Anti-NKB antibody was conjugated with a fluorescent probe (Zenon Antibody Labeling Kit, Molecular Probes) to avoid crossreaction. After rinsing with PBS, sections were incubated with Alexa Fluor 568-conjugated rabbit anti-NKB antibody (1:1000) for 24 h at 4 ◦ C. Confocal images were acquired using a LSM710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). 2.3. Post-embedding double immunoelectron microscopy Animals (n = 3) were perfused with physiological saline, followed by 4% PFA and 0.5% glutaraldehyde in 0.1 M PBS (pH 7.4). Brains were removed and immersed in the same fixative for 2 h, before 50-m coronal sectioning using a microslicer (Dosaka EM, Kyoto, Japan). Sections containing ARC and ME were treated overnight with 0.1 M PBS containing 1% ammonium chloride to quench free aldehydes, then dehydrated in an ascending series of ethanol and embedded in Quetol 812. Ultrathin sections were prepared using an ultramicrotome (EM-UC7, Leica Microsystems) and mounted on 150-mesh nickel grids for immunocytochemistry. The post-embedding method for immunocytochemistry of kisspeptin began with incubation of specimens in 5% normal goat serum for 2 h at RT before incubation with primary antibodies (1:1500) overnight at 4 ◦ C. Next, samples were incubated with goat anti-mouse IgG (H + L) labeled with either 10 or 15 nm colloidal gold for 2 h at RT. For NKB immunocytochemistry, sections were blocked in 10% normal goat serum for 2 h at RT before incubation with primary antibody (1:750) diluted in 0.1 M PBS overnight at 4 ◦ C.
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Fig. 1. Dual immunofluorescence of kisspeptin (red) and NKB (green) (A), kisspeptin (red) and DynA (green) (B), and NKB (red) and DynA (green) (C) in the arcuate nucleus (ARC) of the female rat observed by confocal scanning microscopy. Arrowheads indicate cell bodies co-expressing both peptides (A–C). The dot-like signals indicate the labeling within the fiber and these are not background noises. Scale bars, 50 m.
The following day, samples were incubated with goat anti-rabbit IgG labeled with 5, 10 or 15 nm colloidal gold diluted in 0.1 M PBS at 1:50. For DynA immunocytochemistry, sections were incubated with 10% normal goat serum diluted in 0.1 M PBS for 2 h at RT before incubation with primary antibody (1:300) diluted in 0.1 M PBS for 2 days at 4 ◦ C. Next, specimens were incubated with goat anti-rabbit IgG labeled with either 10 or 15 nm colloidal gold diluted in 0.1 M PB to 1:50. Specimens were rinsed with 0.1 M PB and distilled water, then dried. Finally, samples were stained with 8% uranyl acetate and lead citrate, washed with distilled water and dried. Specimens were observed with a Hitachi H-7650 transmission electron microscope at an accelerating potential of 80 kV. Double immunoelectron microscopy with two different antibodies was performed using the front and back of ultrathin sections mounted on nickel grids. First, immunocytochemistry with primary antibody was performed on one side of the specimen and visualized using one size of colloidal gold particle. Next, the other antibody was visualized on the other side of the specimen using a different size of colloidal gold particle. Immunocytochemical controls were performed in the complete absence of antibodies, without primary specific antibody, and without secondary specific antibody.
3. Results 3.1. Co-localization of kisspeptin, NKB and DynA in the ARC Dual-labeled immunofluorescence for kisspeptin and either NKB or DynA is shown in Fig. 1. Immunoreactivity (ir) within cell bodies and fibers was observed both in the ARC and ME. The majority of kisspeptin-ir neurons were also NKB-ir (Fig. 1A) or DynA-ir (Fig. 1B). Further, NKB-ir neurons were also positive for DynA (Fig. 1C). Thus, within a single neuron, co-localization of kisspeptin, NKB and DynA is present both in cell bodies located in the ARC and fibers projecting to the ME. Subcellular localization of kisspeptin, NKB, or DynA in separate secretory vesicles NKB and kisspeptin localized on different neurosecretory vesicles within axons of the ARC (Fig. 2A). Variation of colloidal gold particle size still resulted in presentation of neuropeptides in separate neurosecretory vesicles (Fig. 2B). Immunoreactivity of DynA and kisspeptin (Fig. 2C and D), or DynA and NKB (Fig. 2E and F) was also observed in individual neurosecretory vesicles located within axons. Although approximately 100–150 axons including immunoreactive neurosecretory vesicles
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Fig. 2. Double immunoelectron micrographs of the axons of KNDy neurons in the ARC. Open arrowheads show kisspeptin-immunoreactive (ir) vesicles. Solid arrowheads, NKB-ir vesicles. Arrows, DynA-ir vesicles. NKB (15 nm colloidal gold) and kisspeptin (10 nm colloidal gold) localized on different neurosecretory vesicles in the axon (A). Immunopositive vesicles under exchange of the size of colloidal gold (B). Kisspeptin and DynA (C and D, using 10 and 15 nm colloidal gold), or DynA and NKB (E and F, using 5 and 15 nm colloidal gold) are distributed on different neurosecretory vesicles. G is lower magnification of E, which shows axon of KNDy neuron. Scale bars, 100 nm.
were observed for each combination, no neurosecretory vesicle containing different sizes of colloids were identified. Similarly, subcellular distribution of kisspeptin, NKB or DynA was confirmed in cell bodies with results similar to those obtained for axons (Fig. 3). Consequently, within axons and cell bodies of KNDy neurons, kisspeptin, NKB and DynA are each packaged into distinct neurosecretory vesicles. 4. Discussion In the present study, immunoelectron microscopy was used to definitively determine subcellular location of kisspeptin, NKB and DynA in KNDy neurons of female rats. All three neuropeptides presented separately both within cell bodies and axons of the external layer of ME. In KNDy neurons, these three neuropeptides are individually sorted and packed into neurosecretory vesicles, suggesting
these neuropeptides are differentially synthesized depending on environmental changes. These results coincide with previous studies, as expression of kisspeptin and NKB are differentially regulated depending on sex difference and sex steroid hormones [26]. Central administration of senktide, an NKB receptor agonist, does not significantly affect Kiss1 expression, but the percentage of Kiss1 neurons expressing c-fos increases with senktide in OVX + E2 female rats [32,33]. Conversely, central administration of nor-BNI, a DynA receptor antagonist, significantly increases LH release only in the presence of negative feedback levels of E2 [27]; this mechanism blocks senktide-dependent LH pulse actions within these rats [32]. Moreover, pretreatment with DynA attenuates senktide-induced activity within KNDy neurons of intact male mice; whereas, it does not alter the average firing frequency pattern activated by senktide in neurons from castrated male mice [25]. Additionally, NK3R is expressed in Kiss1 neurons of mice [5] and sheep [21]; Dyn in
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Fig. 3. Double immunoelectron micrographs of the cell bodies of KNDy neurons in the ARC. Open arrowheads show kisspeptin-ir neurosecretory vesicles, solid arrowheads show NKB-ir vesicles, and arrows show DynA-ir vesicles, respectively. Kisspeptin and NKB (A, 10 and 15 nm colloidal gold), or kisspeptin and DynA (B, 10 and 15 nm colloidal gold), or DynA and NKB (C, 5 and 15 nm colloidal gold) are distributed on different neurosecretory vesicles. (a) and (b) are higher magnifications of the part with the square areas in A, B and C. N, nucleus. Scale bars, 1 m (A–C), 100 nm (a, b).
the neurons of rats [6]; and KOR expression has been identified in Kiss1 neurons of mice [19]. These findings support the hypothesis of KNDy neurons interacting with each other to generate oscillatory communication through extensive collaterals capable of synchronizing comprehensive neurocircuit activity [5,6]. Taken together, it is possible that NKB is synthesized primarily to bring about the onset of kisspeptin activation and, subsequently, preferential synthesis of DynA terminates that action in KNDy neurons. As the majority of kisspeptin neurons located within the ARC do not communicate with GnRH neurons [34], burst firing from each KNDy neuron synchronizes the
interconnected network [4,5,8]. Synthesis of kisspeptin, NKB and DynA is coordinated by physiological changes such as sex steroid hormones, stress or nutritional status. To understand formation of kisspeptin/GnRH/LH pulses, further studies are required to elucidate interactions between kisspeptin, NKB and DynA. The mechanisms underlying sorting and packaging of these neuropeptides to individual neurosecretory vesicles remain unclear. One possibility involves sorting-related peptides, such as secretogranins/chromogranins. Evidence suggests granins are important for regulating sorting and packaging of native peptides or hormones within endocrine or neuroendocrine cells [35]. Investigation into
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expression of granins within KNDy neurons would help to elucidate sorting and packaging of kisspeptin, NKB and DynA. The release mechanism of each vesicle and ratios of individual neuropeptide formation within vesicles also remain unknown. Cellular and molecular biological approaches will be necessary for further investigation. In summary, post-embedding double immunoelectron microscopy revealed subcellular localization patterns of kisspeptin, NKB and DynA within KNDy neurons of female rats. Our results indicate that each of these neuropeptides was contained in individual neurosecretory vesicles, suggesting that synthesis of individual KNDy peptides is differentially regulated. These findings will be clarified by further investigation into specific roles of DynA and NKB on KNDy neurons by autocrine and/or paracrine action in the ARC and regulation of GnRH/LH pulse generation.
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Acknowledgements This study was supported by Grants-in-Aid for Scientific Research (No. 26460323 to H.O. and No. 15K18979 to K.I.) from the Japan Society for the Promotion of Science (JSPS) KAKENHI, and the Ministry of Education, Sports, Science.
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Research paper
Immunoelectron microscopic observation of the subcellular localization of kisspeptin, neurokinin B and dynorphin A in KNDy neurons in the arcuate nucleus of the female rat Hiroko Murakawa a,b , Kinuyo Iwata a , Toshiyuki Takeshita b , Hitoshi Ozawa a,∗ a
Department of Anatomy and Neurology, Graduate School of Medicine, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, Japan Department of Reproductive Medicine, Perinatology and Gynecologic Oncology, Graduate School of Medicine, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, Japan b
h i g h l i g h t s • ARC kisspeptin neurons co-express with NKB and DynA. • Subcellular localization of kisspeptin, NKB and DynA in KNDy neurons were observed by immunoelectron microscopy. • Kisspeptin, NKB and DynA are contained within separate individual neurosecretory vesicles.
a r t i c l e
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Article history: Received 23 October 2015 Received in revised form 25 November 2015 Accepted 4 December 2015 Available online 8 December 2015 Keywords: GnRH/LH pulse KNDy neuron Immunoelectron microscopy Neurosecretory vesicle Reproduction
a b s t r a c t KNDy neurons are named for their co-expression of three neuropeptides, kisspeptin, neurokinin B (NKB) and dynorphin A (DynA). These cells, located in the hypothalamic arcuate nucleus (ARC), are associated with generation of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulses to control follicular growth and steroidogenesis. However, subcellular sorting mechanisms for secretory vesicles containing these neuropeptides have not been elucidated. In this study, we analyzed the localization pattern of kisspeptin, NKB and DynA in the ARC of the ovariectomized rat immediately treated with estrogen using immunoelectron microscopy. First, we identified neuropeptides by dual-labeled fluorescence immunohistochemistry, with results indicating all three neuropeptides co-express within individual ARC cells in female rats. Next, we investigated the subcellular localization pattern of kisspeptin, NKB, and/or DynA using post-embedding double immunoelectron microscopy, indicating that each type of neuropeptide is contained within separate and individual neurosecretory vesicles. This suggests sorting and packaging of kisspeptin, NKB and DynA is differentially regulated within KNDy neurons. Our findings facilitate understanding of regulatory mechanisms underlying kisspeptin secretion in KNDy neurons, and generation of GnRH/LH pulses induced by kisspeptin in the ARC. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) pulses are necessary for reproduction [1]. Abnormal LH pulses have been observed in women with polycystic ovary syndrome (PCOS) [2] and weight loss-related amenorrhea [3]. Thus, GnRH/LH
∗ Corresponding author at: Department of Anatomy and Neurobiology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 1138602, Japan. Fax: +81 3 5685 6640. E-mail address: [email protected] (H. Ozawa). http://dx.doi.org/10.1016/j.neulet.2015.12.008 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
pulse abnormalities have the potential to cause menstrual disorders, anovulation and infertility. Recent studies suggest pulsatile GnRH/LH secretion is generated by KNDy neurons [4]. These cells, which co-express kisspeptin, neurokinin B (NKB) and dynorphin A (DynA), have been identified in the hypothalamic arcuate nucleus (ARC) of mice [5], rats [6], ewes [7] and goats [8]. Kisspeptin fibers project, in a non-synaptic manner, to GnRH terminals located within the median eminence (ME) of monkeys [9], rats and goats [10,11]. GnRH neurons expressing kisspeptin receptors (Kiss1r) have been identified in mice [12] and rats [13]. KNDy neurons also express estrogen receptor ␣ (ER␣)
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in mice [14], rats [6,15] and ewes [16], and receive to negative feedback of estrogen to regulate LH release [14,15]. NKB also induces LH release, as evidenced by the ability of a selective NKB receptor (NK3R) agonist, senktide, to increase LH secretion in sheep [17] and multiple-unit electrical activity (MUA) volleys in the ARC of goats [8]. In contrast, DynA inhibits LH release, as demonstrated by the capacity of a selective DynA receptor (KOR) antagonist, nor-binaltorphimine (nor-BNI), to increase LH pulse frequency in sheep [18]. Further, MUA volleys in the ARC are inhibited by DynA and increased by nor-BNI in goats [8]. As KNDy fibers have been observed in close apposition to KNDy cell bodies in the ARC of rats [6], ovine [19] and goats [8], it is likely KNDy neurons communicate with each other. KNDy neurons express NK3R in mice [5], rats [6,20], and sheep [21]; KOR in mice [5]; but, do not express Kiss1r in mice [22] and ovine [23]. In rats, GnRH neurons do not express KOR and only slightly express NK3R [20,24]; while in sheep, these cells do not express NK3R [21]. Further, as senktide was shown to have no effect on GnRH neuronal firing in castrated male mice [25], GnRH neurons appear to be unaffected by NKB or DynA. Thus, KNDy neurons are thought to act as GnRH/LH pulse generators within neural circuits by regulating kisspeptin secretion through stimulation by NKB/NK3R signaling and inhibition by DynA/KOR signaling. Though co-expression of kisspeptin, NKB and DynA has previously been reported in mice [5], ewes [7] and goats [8], studies examining rat KNDy neurons indicate only proNKB and prodynorphin co-localize within ARC kisspeptin neurons [6]. Moreover, subcellular localization of neuropeptides within these cells has not been elucidated. Thus, in the current study, we investigated the subcellular localization of kisspeptin, NKB and DynA within KNDy neurons of female rats by double immunoelectron microscopy. 2. Materials and methods 2.1. Animals Adult Wistar-Imamichi strain female rats, weighing 200–250 g, aged 9–14 weeks (Institute for Animal Reproduction, Ibaraki, Japan), were housed in an air-conditioned room (23 ± 1 ◦ C) under 14 h light (6:00–20:00)/10 h dark (20:00–6:00) cycles with access to food and water. Timing of estrous cycle was monitored by daily vaginal smear cytology. Animals demonstrating at least two consecutive, regular 4-day estrous cycles were used in subsequent experiments. Animals were ovariectomized (OVX) and immediately received a Silastic tube implant filled with 20 g/ml -estradiol (E2; Sigma–Aldrich, St. Louis, MO) dissolved in sesame oil, for 7–13 days. As kisspeptin-immunoreactive (ir) and NKB-ir areas in the ARC are more prevalent in OVX + low E2 rats compared with OVX rats [26,27], and preliminary studies indicated DynA-ir areas in OVX + E2 rats were also larger than that of OVX rats, E2 implants were intended to yield plasma E2 levels similar to diestrus [28]. All surgeries were conducted under inhalational anesthesia using isoflurane. For perfusion fixation, animals were deeply anesthetized with sodium pentobarbital (50 mg/kg). Experimental procedures were conducted in accordance with Nippon Medical School’s Guidance on Animal Bioethics (based on guidelines for humane treatment of experimental animals issued by the US National Institutes of Health) and with approval from Nippon Medical School’s Committee on the Care and Use of Experimental Animals. 2.2. Antibodies Primary antibodies included mouse monoclonal anti-kisspeptin antibody (kindly provided by Dr. Ohtaki, Takeda Pharmaceutical
Co., Osaka, Japan) [29], rabbit polyclonal anti-NKB antibody (Novus Biological, Littleton, CO) [30], and rabbit anti-dynorphin A antibody (Phoenix Pharmaceuticals, Inc. Burlingame, CA) [31]. For fluorescent immunohistochemistry, secondary antibodies included Alexa Fluor 568-conjugated donkey anti-mouse immunoglobulin (IgG) (dilution 1:500; Molecular Probes, Eugene, OR), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:500; Molecular Probes), and Alexa Fluor 568-conjugated antirabbit IgG (Zenon kit, Molecular Probes). For post-embedding immunoelectron microscopy, secondary antibodies included goat anti-rabbit IgG labeled with 5 or 15 nm colloidal gold (BB International, Cardiff, UK), goat F(ab)2 anti-rabbit IgG labeled with 10 nm colloidal gold (BB International) and goat anti-mouse IgG (H + L) labeled with 10 or 15 nm colloidal gold (BB International), used at a dilution of 1:50. Dual-labeled fluorescence immunohistochemistry for kisspeptin and NKB, kisspeptin and DynA, or NKB and DynA in the ARC. Animals (n = 2) were perfused through the heart with physiological saline, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS) (pH 7.4). Brains were removed, post-fixed for 24 h in the same fixative at 4 ◦ C, then cryoprotected in 0.1 M PBS containing 30% sucrose and 0.9% NaCl at 4 ◦ C. Using a cryostat, brains were cut into 40-m-thick coronal sections (CM3050S, Leica Microsystems, Wezlar, Germany) before storage in cryoprotectant solution at −20 ◦ C until use in immunofluorescence studies. To perform double labeling for kisspeptin and either NKB or DynA, sections were incubated with primary mouse anti-kisspeptin antibody (1:5000) and either rabbit anti-NKB antibody (1:5000) or rabbit anti-DynA antibody (1:5000) for 4 days at 4 ◦ C. After they had been washed with PBS, sections were incubated with Alexa Fluor 568-conjugated donkey anti-mouse IgG and Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibodies for 2 h at room temperature (RT). To perform double labeling for DynA and NKB, sections were incubated with rabbit anti-DynA antibody (1:5000) at 4 ◦ C for 4 days. Sections were rinsed with PBS and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1:500) for 2 h at RT. Anti-NKB antibody was conjugated with a fluorescent probe (Zenon Antibody Labeling Kit, Molecular Probes) to avoid crossreaction. After rinsing with PBS, sections were incubated with Alexa Fluor 568-conjugated rabbit anti-NKB antibody (1:1000) for 24 h at 4 ◦ C. Confocal images were acquired using a LSM710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). 2.3. Post-embedding double immunoelectron microscopy Animals (n = 3) were perfused with physiological saline, followed by 4% PFA and 0.5% glutaraldehyde in 0.1 M PBS (pH 7.4). Brains were removed and immersed in the same fixative for 2 h, before 50-m coronal sectioning using a microslicer (Dosaka EM, Kyoto, Japan). Sections containing ARC and ME were treated overnight with 0.1 M PBS containing 1% ammonium chloride to quench free aldehydes, then dehydrated in an ascending series of ethanol and embedded in Quetol 812. Ultrathin sections were prepared using an ultramicrotome (EM-UC7, Leica Microsystems) and mounted on 150-mesh nickel grids for immunocytochemistry. The post-embedding method for immunocytochemistry of kisspeptin began with incubation of specimens in 5% normal goat serum for 2 h at RT before incubation with primary antibodies (1:1500) overnight at 4 ◦ C. Next, samples were incubated with goat anti-mouse IgG (H + L) labeled with either 10 or 15 nm colloidal gold for 2 h at RT. For NKB immunocytochemistry, sections were blocked in 10% normal goat serum for 2 h at RT before incubation with primary antibody (1:750) diluted in 0.1 M PBS overnight at 4 ◦ C.
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Fig. 1. Dual immunofluorescence of kisspeptin (red) and NKB (green) (A), kisspeptin (red) and DynA (green) (B), and NKB (red) and DynA (green) (C) in the arcuate nucleus (ARC) of the female rat observed by confocal scanning microscopy. Arrowheads indicate cell bodies co-expressing both peptides (A–C). The dot-like signals indicate the labeling within the fiber and these are not background noises. Scale bars, 50 m.
The following day, samples were incubated with goat anti-rabbit IgG labeled with 5, 10 or 15 nm colloidal gold diluted in 0.1 M PBS at 1:50. For DynA immunocytochemistry, sections were incubated with 10% normal goat serum diluted in 0.1 M PBS for 2 h at RT before incubation with primary antibody (1:300) diluted in 0.1 M PBS for 2 days at 4 ◦ C. Next, specimens were incubated with goat anti-rabbit IgG labeled with either 10 or 15 nm colloidal gold diluted in 0.1 M PB to 1:50. Specimens were rinsed with 0.1 M PB and distilled water, then dried. Finally, samples were stained with 8% uranyl acetate and lead citrate, washed with distilled water and dried. Specimens were observed with a Hitachi H-7650 transmission electron microscope at an accelerating potential of 80 kV. Double immunoelectron microscopy with two different antibodies was performed using the front and back of ultrathin sections mounted on nickel grids. First, immunocytochemistry with primary antibody was performed on one side of the specimen and visualized using one size of colloidal gold particle. Next, the other antibody was visualized on the other side of the specimen using a different size of colloidal gold particle. Immunocytochemical controls were performed in the complete absence of antibodies, without primary specific antibody, and without secondary specific antibody.
3. Results 3.1. Co-localization of kisspeptin, NKB and DynA in the ARC Dual-labeled immunofluorescence for kisspeptin and either NKB or DynA is shown in Fig. 1. Immunoreactivity (ir) within cell bodies and fibers was observed both in the ARC and ME. The majority of kisspeptin-ir neurons were also NKB-ir (Fig. 1A) or DynA-ir (Fig. 1B). Further, NKB-ir neurons were also positive for DynA (Fig. 1C). Thus, within a single neuron, co-localization of kisspeptin, NKB and DynA is present both in cell bodies located in the ARC and fibers projecting to the ME. Subcellular localization of kisspeptin, NKB, or DynA in separate secretory vesicles NKB and kisspeptin localized on different neurosecretory vesicles within axons of the ARC (Fig. 2A). Variation of colloidal gold particle size still resulted in presentation of neuropeptides in separate neurosecretory vesicles (Fig. 2B). Immunoreactivity of DynA and kisspeptin (Fig. 2C and D), or DynA and NKB (Fig. 2E and F) was also observed in individual neurosecretory vesicles located within axons. Although approximately 100–150 axons including immunoreactive neurosecretory vesicles
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Fig. 2. Double immunoelectron micrographs of the axons of KNDy neurons in the ARC. Open arrowheads show kisspeptin-immunoreactive (ir) vesicles. Solid arrowheads, NKB-ir vesicles. Arrows, DynA-ir vesicles. NKB (15 nm colloidal gold) and kisspeptin (10 nm colloidal gold) localized on different neurosecretory vesicles in the axon (A). Immunopositive vesicles under exchange of the size of colloidal gold (B). Kisspeptin and DynA (C and D, using 10 and 15 nm colloidal gold), or DynA and NKB (E and F, using 5 and 15 nm colloidal gold) are distributed on different neurosecretory vesicles. G is lower magnification of E, which shows axon of KNDy neuron. Scale bars, 100 nm.
were observed for each combination, no neurosecretory vesicle containing different sizes of colloids were identified. Similarly, subcellular distribution of kisspeptin, NKB or DynA was confirmed in cell bodies with results similar to those obtained for axons (Fig. 3). Consequently, within axons and cell bodies of KNDy neurons, kisspeptin, NKB and DynA are each packaged into distinct neurosecretory vesicles. 4. Discussion In the present study, immunoelectron microscopy was used to definitively determine subcellular location of kisspeptin, NKB and DynA in KNDy neurons of female rats. All three neuropeptides presented separately both within cell bodies and axons of the external layer of ME. In KNDy neurons, these three neuropeptides are individually sorted and packed into neurosecretory vesicles, suggesting
these neuropeptides are differentially synthesized depending on environmental changes. These results coincide with previous studies, as expression of kisspeptin and NKB are differentially regulated depending on sex difference and sex steroid hormones [26]. Central administration of senktide, an NKB receptor agonist, does not significantly affect Kiss1 expression, but the percentage of Kiss1 neurons expressing c-fos increases with senktide in OVX + E2 female rats [32,33]. Conversely, central administration of nor-BNI, a DynA receptor antagonist, significantly increases LH release only in the presence of negative feedback levels of E2 [27]; this mechanism blocks senktide-dependent LH pulse actions within these rats [32]. Moreover, pretreatment with DynA attenuates senktide-induced activity within KNDy neurons of intact male mice; whereas, it does not alter the average firing frequency pattern activated by senktide in neurons from castrated male mice [25]. Additionally, NK3R is expressed in Kiss1 neurons of mice [5] and sheep [21]; Dyn in
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Fig. 3. Double immunoelectron micrographs of the cell bodies of KNDy neurons in the ARC. Open arrowheads show kisspeptin-ir neurosecretory vesicles, solid arrowheads show NKB-ir vesicles, and arrows show DynA-ir vesicles, respectively. Kisspeptin and NKB (A, 10 and 15 nm colloidal gold), or kisspeptin and DynA (B, 10 and 15 nm colloidal gold), or DynA and NKB (C, 5 and 15 nm colloidal gold) are distributed on different neurosecretory vesicles. (a) and (b) are higher magnifications of the part with the square areas in A, B and C. N, nucleus. Scale bars, 1 m (A–C), 100 nm (a, b).
the neurons of rats [6]; and KOR expression has been identified in Kiss1 neurons of mice [19]. These findings support the hypothesis of KNDy neurons interacting with each other to generate oscillatory communication through extensive collaterals capable of synchronizing comprehensive neurocircuit activity [5,6]. Taken together, it is possible that NKB is synthesized primarily to bring about the onset of kisspeptin activation and, subsequently, preferential synthesis of DynA terminates that action in KNDy neurons. As the majority of kisspeptin neurons located within the ARC do not communicate with GnRH neurons [34], burst firing from each KNDy neuron synchronizes the
interconnected network [4,5,8]. Synthesis of kisspeptin, NKB and DynA is coordinated by physiological changes such as sex steroid hormones, stress or nutritional status. To understand formation of kisspeptin/GnRH/LH pulses, further studies are required to elucidate interactions between kisspeptin, NKB and DynA. The mechanisms underlying sorting and packaging of these neuropeptides to individual neurosecretory vesicles remain unclear. One possibility involves sorting-related peptides, such as secretogranins/chromogranins. Evidence suggests granins are important for regulating sorting and packaging of native peptides or hormones within endocrine or neuroendocrine cells [35]. Investigation into
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expression of granins within KNDy neurons would help to elucidate sorting and packaging of kisspeptin, NKB and DynA. The release mechanism of each vesicle and ratios of individual neuropeptide formation within vesicles also remain unknown. Cellular and molecular biological approaches will be necessary for further investigation. In summary, post-embedding double immunoelectron microscopy revealed subcellular localization patterns of kisspeptin, NKB and DynA within KNDy neurons of female rats. Our results indicate that each of these neuropeptides was contained in individual neurosecretory vesicles, suggesting that synthesis of individual KNDy peptides is differentially regulated. These findings will be clarified by further investigation into specific roles of DynA and NKB on KNDy neurons by autocrine and/or paracrine action in the ARC and regulation of GnRH/LH pulse generation.
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Acknowledgements This study was supported by Grants-in-Aid for Scientific Research (No. 26460323 to H.O. and No. 15K18979 to K.I.) from the Japan Society for the Promotion of Science (JSPS) KAKENHI, and the Ministry of Education, Sports, Science.
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