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The Journal of Reproduction and Development logoLink to The Journal of Reproduction and Development
. 2025 Jan 27;71(2):71–84. doi: 10.1262/jrd.2024-100

Involvement of nuclear receptor corepressor 2 (NCOR2) in estrogen-induced repression of arcuate Kiss1 expression in female rats

Marina TAKIZAWA 1, Sae MIYAZAKI 1, Hitomi TSUCHIDA 1, Mayuko NAGAE 1, Shunsuke SEKI 1, Masumi HIRABAYASHI 2, Fumitaka OSAKADA 3, Naoko INOUE 1, Hiroko TSUKAMURA 1, Yoshihisa UENOYAMA 1
PMCID: PMC11999826  PMID: 39864859

Abstract

Hypothalamic arcuate (ARC) kisspeptin neurons are considered the gonadotropin-releasing hormone pulse generator in rats. In virgin rats, the expression of the ARC kisspeptin gene (Kiss1) is repressed by proestrous levels of estradiol-17β (high E2) but not by diestrous levels of E2 (low E2). In lactating rats, ARC Kiss1 expression is repressed by low E2 during late lactation. This study aimed to investigate whether nuclear receptor corepressor 2 (NCOR2, encoded by Ncor2), an estrogen receptor α corepressor, is involved in the estrogen-induced repression of ARC Kiss1 expression in rats. Double in situ hybridization for Kiss1 and Ncor2 revealed that approximately 80% of ARC Kiss1-expressing cells co-expressed Ncor2 in ovariectomized (OVX) + low E2 virgin rats, while approximately 90% of ARC Kiss1-expressing cells co-expressed Ncor2 in OVX + low E2 lactating rats. To further examine the role of Ncor2, we studied the effects of Kiss1-dependent Ncor2 knockdown on ARC Kiss1 expression and luteinizing hormone (LH) pulses. An adeno-associated virus vector carrying Cre-activated short hairpin RNA (shRNA) for Ncor2 was administered to the ARC in two Kiss1-Cre rat models: OVX + high E2 Kiss1-Cre virgin rats and OVX + low E2 Kiss1-Cre lactating rats. Ncor2-shRNA treatment significantly increased the number of ARC Kiss1-expressing cells and the intensity of Kiss1 signals in OVX + high E2 virgin rats but failed to fully restore low E2-induced Kiss1 repression in lactating rats. The Ncor2-shRNA treatment failed to affect LH pulses in both models. These findings suggest that NCOR2 in ARC kisspeptin neurons mediates high E2-induced repression of ARC Kiss1 expression in virgin rats.

Keywords: Estrogen negative feedback, Kisspeptin, Lactation, NCOR2, Reproduction

Accumulating evidence suggests that hypothalamic kisspeptin neurons are master regulators of reproduction via direct stimulation of gonadotropin-releasing hormone (GnRH) and gonadotropin release in mammalian species, such as rodents [1,2,3,4], ruminants [5,6,7], non-human primates [8,9,10], and humans [11]. Specifically, kisspeptin neurons located in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV)/preoptic area (POA) are suggested to serve as GnRH pulse and surge generators, respectively, in female rodents [12,13,14,15,16], pigs [17], ruminants [18, 19], and primates [8, 9, 11]. Importantly, a majority of ARC and AVPV kisspeptin neurons express estrogen receptor α (ERα) in rodents [3, 12, 20, 21] and ruminants [22]. Kisspeptin gene (Kiss1) expression in the ARC is downregulated by circulating estrogen; thus, the ARC Kiss1 expression level is lowest at the proestrous stage and highest at the diestrous stage in ovary-intact female rodents [3, 12, 20, 23]. Therefore, ARC kisspeptin neurons act as sites of estrogen-negative feedback to suppress GnRH/luteinizing hormone (LH) pulses. Notably, proestrous levels of estradiol-17β (high E2) strongly repress ARC Kiss1 expression; however, diestrous levels of E2 (low E2) fail to repress the expression in ovariectomized (OVX) rats [12]. Furthermore, in lactating rats, low E2 represses ARC Kiss1 and LH pulses during late lactation (later than 10 days after parturition during the 20 days of lactation) [24]. Contrary to ARC kisspeptin neurons, AVPV/POA kisspeptin neurons act as sites of estrogen-positive feedback to trigger GnRH/LH surges and consequent ovulation in rodents and primates because AVPV/POA Kiss1 expression is upregulated by estrogen in rodents [12, 20, 21, 25] and Japanese monkeys [26]. ERα mediates the estrogen-induced downregulation and upregulation of Kiss1 expression in kisspeptin neurons in the ARC and AVPV, respectively [12, 20, 21, 27], because estrogen failed to repress or enhance Kiss1 expression in the ARC or AVPV, respectively, in OVX Esr1 (encoding ERα) knockout mice [20]. The ERα-dependent repression or activation of ERα target genes is often mediated by ERα coregulators, such as corepressors or coactivators for the transcriptional activity of ERα [28, 29].

In the present study, we found that the nuclear receptor corepressor 2 (NCOR2) gene (Ncor2), an ERα corepressor gene, was highly expressed in the ARC kisspeptin neurons using RNA-seq data of isolated visualized kisspeptin neurons obtained from the ARC of female Kiss1-tdTomato heterozygous rats [30]. NCOR2 (also known as SMRT, which stands for silencing mediator of retinoic acid and thyroid hormone receptor) is a classic nuclear receptor corepressor that binds to ERα and serves as an adaptor to recruit proteins possessing histone deacetylase (HDAC) activity, which is known to repress gene expression by maintaining chromatin in a more condensed state [28, 29]. Notably, our previous study demonstrated that high E2 decreases histone acetylation of the Kiss1 promoter region in the ARC of female mice, whose ARC Kiss1 expression is strongly repressed, and that inhibition of histone deacetylation enhances in vitroKiss1 expression in non-Kiss1-expressing immortalized mouse cell lines [31]. Therefore, NCOR2 may be involved in high E2-induced repression of ARC Kiss1 expression in rodents. In contrast, in lactating rats, low E2 is sufficient to repress ARC Kiss1 expression and LH pulses during late lactation, as ARC Kiss1 expression and LH pulses were suppressed in ovary-intact or low E2-treated OVX lactating rats but not in OVX lactating rats during late lactation [24]. These findings suggest that the high E2-induced repression of ARC Kiss1 expression in cyclic rats and low E2-dependent lactation-induced repression of ARC Kiss1 expression may differ in terms of the involvement of NCOR2.

The present study aimed to investigate whether NCOR2 is involved in the estrogen-induced repression of ARC Kiss1 expression using two rat models: proestrous model rats that were OVX and treated with high E2, and OVX lactating rats treated with low E2. We first examined the co-expression of the Ncor2 in ARC and AVPV kisspeptin neurons using double in situ hybridization (ISH) for Ncor2 and Kiss1 in OVX wild-type virgin and lactating rats treated with low E2. We examined the effects of Kiss1-dependent Ncor2 knockdown on ARC Kiss1 expression and pulsatile LH release by introducing an adeno-associated virus (AAV) vector carrying Cre-activated short hairpin RNA (shRNA) against Ncor2 into the ARC of OVX Kiss1-Cre virgin rats treated with high E2 and OVX Kiss1-Cre lactating rats treated with low E2 during late lactation, during which Kiss1 and LH pulses are largely suppressed [12, 24].

Materials and Methods

Animals

Kiss1-tdTomato heterozygous female rats [4], wild-type Wistar–Imamichi female rats (Institute for Animal Reproduction, Kasumigaura, Japan), and Kiss1-Cre female rats [32] were used in this study. Kiss1-tdTomato heterozygous and Kiss1-Cre rats were raised in our laboratory and validated in our previous studies [4, 32]. Animals were maintained in a room with a 14/10 h light/dark cycle (lights on 0500 h) at 22 ± 3°C and had free access to food (CE-2; CLEA Japan, Tokyo, Japan) and water. Animals (8–13 weeks of age) with two consecutive regular 4-day estrous cycles, as determined by daily observation of vaginal smears, were used. Kiss1-tdTomato heterozygous female rats, whose coding sequences for Kiss1 exons 2 and 3 were replaced with the tdTomato gene, were subjected to RNA-seq analysis, as described previously [30]. Wild-type Wistar–Imamichi female rats were subjected to RT-PCR analysis to examine ARC Ncor2 expression and histological analysis to determine Ncor2 expression in Kiss1-expressing cells in the ARC and AVPV. Kiss1-Cre female rats, whose coding sequences for Kiss1 exons 2 and 3 were replaced with Cre recombinase [32], were used for Kiss1-dependent Ncor2 knockdown in the ARC.

Kiss1-tdTomato and wild-type female rats were bilaterally OVX, and some were treated with low or high E2. Briefly, the animals were OVX for 2 weeks to serve as the OVX group. Some OVX rats were immediately treated with a subcutaneous Silastic tubing (1.57 mm inner diameter; 3.18 mm outer diameter; 25 mm in length; Dow Corning, Midland, MI, USA) filled with E2 (Sigma-Aldrich, St. Louis, MO, USA), dissolved in peanut oil (Sigma-Aldrich) at 20 µg/ml, for 1 week to serve as OVX + low E2 rats, in which plasma E2 levels mimicked the diestrous level [33]. Some OVX rats were treated with a subcutaneous Silastic tubing filled with low E2 for 5 days, followed by a Silastic tubing (1.57 mm inner diameter; 3.18 mm outer diameter; 28 mm in length) filled with high E2, dissolved in peanut oil at 1,000 µg/ml, for 2 days to serve as the OVX + high E2 rats, in which plasma E2 levels mimicked the proestrous level [34].

Wild-type or Kiss1-Cre lactating rats were obtained by mating with a male overnight on the day of proestrus, and pregnant females were housed individually. The day of parturition was designated day 0 postpartum, and the litter size of the lactating rats was adjusted to eight (four males and four females) on day 1 postpartum. Non-lactating rats were obtained by depriving all pups on day 1 postpartum. On day 2 postpartum, all animals were bilaterally OVX, and some OVX lactating rats were immediately implanted with a Silastic tubing containing low E2. The E2-containing tubing was replaced with new tubing on day 9 postpartum to maintain a constant plasma E2 level.

The surgical operations were performed under anesthesia with an intraperitoneal injection of ketamine (27 mg/kg; Fujita, Tokyo, Japan) and xylazine (5.3 mg/kg; Bayer AG, Leverkusen, Germany) mixture, followed by inhalation of isoflurane (1–3%, Pfizer Japan, Tokyo, Japan). The care of animals and all experimental procedures performed in the present study were reviewed and approved by the Animal Experiment Committee of Nagoya University.

RNA‑seq analysis of ERα corepressor candidates in isolated tdTomato-positive cells obtained from Kiss1‑tdTomato heterozygous female rats

The expression profiles of ERα corepressor genes in ARC or AVPV kisspeptin neurons were analyzed using RNA-seq data obtained from Kiss1-tdTomato heterozygous rats, as described in our previous study [30]. Briefly, ARC and AVPV tissues were dissected from OVX and OVX + high E2 Kiss1-tdTomato heterozygous rats, respectively, because tdTomato signals were visible in the ARC under the OVX condition and in the AVPV under the OVX + high E2 condition. The tdTomato-positive cells were isolated and picked under a fluorescence microscope using a glass pipette. Ten (for ARC) and three (for AVPV) tdTomato-positive cells were pooled and subjected to RNA-seq analysis. mRNA expression levels were normalized by calculating the reads per kilobase per million mapped reads for each mRNA [30].

Quantitative RT-PCR analysis for Ncor2 mRNA expression levels in the ARC of virgin female rats

To examine the effects of E2 on ARC Ncor2 mRNA expression, wild-type OVX, OVX + low E2, and OVX + high E2 rats (n = 5 in each group) were decapitated between 1300 and 1500 h, as described previously [34], and Ncor2 mRNA levels in the ARC of each group were quantified by RT-PCR. Briefly, the ARC-median eminence (ME) region was dissected from the brain using a microblade, and DNA-free total RNA was purified from the ARC-ME tissue using ISOGEN (Nippon Gene, Tokyo, Japan). The High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to synthesize full-length cDNAs. The obtained cDNA was used as a template in PCR reactions using primers for Ncor2 (5'-ctctcgctgtggaccctaag-3' and 5'-cctgctgatggtacccttgt-3') and Actb (5'-tgtcaccaactgggacgata-3' and 5'-ggggtgttgaaggtctcaaa-3'). Quantitative RT-PCR analysis of Ncor2 mRNA expression was performed using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) with THUNDERBIRD qPCR Mix (TOYOBO, Osaka, Japan), as described previously [34]. The expression levels of Ncor2 mRNA were normalized to those of Actb mRNA.

Histological analysis of co-expression of Ncor2 in the hypothalamic kisspeptin neurons of OVX + low E2 virgin and lactating rats

Free-floating double ISH for Kiss1 and Ncor2 was performed using the hypothalamic sections, including the ARC (from 1.72 to 4.36 mm posterior to the bregma, every fourth section, 13 sections in total) or the AVPV (from 0.12 mm anterior to 0.60 mm posterior to the bregma, every second section, eight sections in total) of wild-type virgin OVX + low E2 rats, as previously described [30, 35, 36]. OVX + low E2 virgin rats were selected because OVX + low E2 rats have been confirmed to show Kiss1 expression in both the ARC and AVPV [12, 30, 34]. Briefly, the animals were perfused with phosphate-buffered saline (PBS) and then with 4% paraformaldehyde (Sigma-Aldrich) in 0.05 M phosphate buffer, and the brains were dissociated and post-fixed with the same fixative overnight. The fixative was replaced with 30% sucrose in 0.05 M phosphate buffer, and brain sections were made by a cryostat (CM1800, Leica Biosystems, Wetzlar, Germany). The sections were hybridized overnight at 60°C with 1 µg/ml fluorescein isothiocyanate (FITC)-labeled Kiss1 anti-sense cRNA probe (position 33-348, AY196983) [37] and digoxigenin (DIG)-labeled Ncor2 anti-sense cRNA probe (position 7196-8366, NM_001108334.1). The FITC-labeled Kiss1 probe on the sections was visualized by peroxidase (POD)-conjugated anti-FITC antibody (RRID: AB_840257, 1:1000, Roche Diagnostics, Basel, Switzerland) and tyramide signal amplification (TSA) Plus FITC Kit (1:100; PerkinElmer, Shelton, CT, USA). After the POD inactivation on the POD-conjugated anti-FITC antibody with 0.1 N hydrochloric acid for 30 min, the DIG-labeled Ncor2 probe on the sections was visualized by a POD-conjugated anti-DIG antibody (RRID: AB_514500, 1:1000, Roche Diagnostics), TSA Plus Biotin Kit (1:100; PerkinElmer), and DyLight 594-conjugated streptavidin (1:500, Thermo Fisher Scientific). No positive signals for Ncor2 mRNA were detected in brain sections hybridized with the corresponding sense probe as a negative control (data not shown). The sections were mounted on slides, and fluorescent images were obtained under a fluorescence microscope with ApoTome2 optical sectioning (Carl Zeiss, Oberkochen, Germany). Kiss1- and Ncor2-positive cells were counted unilaterally on the photomicrographs, according to the rat brain atlas [38].

Free-floating double ISH for Kiss1/Ncor2 or Tac3 (neurokinin B gene)/Ncor2 was performed as described above using hypothalamic sections including the ARC of OVX + low E2 lactating rats. Brains were collected on day 16 postpartum according to our previous study, which showed that ARC Kiss1 expression was repressed in lactating rats in a low E2-dependent manner during late lactation [24]. Tac3 mRNA was used as an indicator of ARC kisspeptin neurons in lactating rats because the majority of ARC kisspeptin neurons co-express neurokinin B in rodents and ruminants [39,40,41], and Tac3 mRNA was comparably expressed in lactating and non-lactating rats on day 8 postpartum [42]. The sections were hybridized with FITC-labeled Kiss1 or Tac3 anti-sense cRNA probe (position 180–483, NM_019162) [43] and DIG-labeled Ncor2 anti-sense cRNA probe. The hybridized probes were detected and analyzed as described above.

In vitro validation of Ncor2-siRNA candidates to repress Ncor2 mRNA expression and NCOR2 protein biosynthesis using mHypoA-55 cell line, a model for rodent ARC kisspeptin neurons

Two candidate sequences of Ncor2-siRNA (anti-sense sequences, including the overhang; siRNA #1, AGACUUUGGUUCCAAAUGCgg; siRNA #2, UUAACUCAUUGAAGGUAUCaa) were designed using siDirect version 2.0 (http://sidirect2.rnai.jp/) with the rat Ncor2 mRNA sequence. These candidate sequences were compatible with rat and mouse Ncor2 mRNA and were matched to generate shRNA. Additionally, we selected two Ncor2-siRNA (siRNA #3, UUAGUUAAGGCUUUAGACAgg; siRNA #4, AUUUACCCAUGAGUGCCUUuc) as previously reported [44]. The four Ncor2-siRNA candidates were synthesized and duplexed by Sigma-Aldrich. The MISSION siRNA Universal Negative Control #1 (siRNA-NC, Sigma-Aldrich) was used as a negative control for in vitro validation of Ncor2-siRNA candidates.

Immortalized mouse neuronal cells, mHypoA-55 cells (RRID: CVCL_D416; CELLutions Biosystems, Burlington, ON, Canada), were used as a model for rodent ARC kisspeptin neurons [34] to validate the silencing effect of Ncor2-siRNA candidates in vitro, as described in our previous study [34]. One of four Ncor2-siRNA candidates or siRNA-NC (10 nM) was transfected into mHypoA-55 cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific). After transfection, the cells were cultured for another 24 or 48 h. The cells were treated with E2 (100 nM) for 2 h and then harvested for the analysis of Ncor2 mRNA levels (n = 4). Total RNA was purified from harvested cells using ISOGEN. ReverTra Ace (TOYOBO, Osaka, Japan) was used to synthesize full-length cDNAs, as described previously [34]. The obtained cDNA was used as a template in PCR reactions using primers for Ncor2 (5'-ctgggacggaaatcttcaac-3' and 5'-ggcattcagagggttaaaagc-3') and Actb (5'-ggtgggaatgggtcagaagg-3' and 5'-gtacatggctggggtgttga-3'). The relative expression levels of Ncor2 mRNA were normalized to those of Actb mRNA.

The siRNA #1, siRNA #2, or siRNA-NC were transfected into the mHypoA-55 cells in a similar manner. Whole-cell lysates were collected 48 h after transfection for analysis of NCOR2 protein levels (n = 4). The lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (GE Healthcare, Chicago, IL, USA), as previously described [34]. The membranes were incubated with rabbit anti-human SMRT polyclonal antibody (RRID: AB_310286; 1:2000, Millipore, Burlington, MA, USA) at 4°C for 16 h. The specificity of the antibody has been confirmed previously [45]. The membranes were then incubated with horseradish POD-conjugated goat anti-rabbit IgG antibody (RRID: AB_955447; 1:10000, Abcam, Cambridge, UK) at room temperature for 1 h. Chemiluminescence was visualized using ECL Prime Western Blotting Detection Reagent (GE Healthcare) and recorded with Light-Capture II (AE-6982; ATTO, Tokyo, Japan). The membranes were treated with Restore PLUS Western Blot Stripping Buffer (Thermo Fisher Scientific) and incubated with rabbit anti-human β-actin polyclonal antibody (RRID: AB_2305186, 1:10000; Abcam, Cambridge, UK). The expression of the β-actin protein was detected using the method described above. Band intensities were estimated by densitometry using ImageJ version 1.53k (https://imagej.nih.gov/ij/). The relative expression levels of NCOR2 protein were normalized by the levels of β-actin protein.

Preparation of AAV carrying Ncor2 shRNA

AAV-DJ vectors carrying shRNA #1 against Ncor2 mRNA were prepared, as previously described [46]. DNA constructs encoding Cre-dependent Ncor2-targeted shRNA #1 or its scrambled-shRNA were designed (Table 1) and synthesized in the pMK plasmid using the GeneArt Strings DNA Fragments service (Thermo Fisher Scientific). The pMK plasmid backbone was replaced with the AAV-U6-EGFP plasmid (Vector Biolabs, Malvern, PA, USA). The AAV-U6-EGFP vector contains an EGFP-expressing sequence under the control of a cytomegalovirus (CMV) promoter. The loop sequence, including the floxed stop cassette in the DNA constructs, was obtained from a previous study [47]. The resulting AAV-U6-shRNA expressed shRNA under the control of the U6 promoter, an RNA polymerase III (Pol III) promoter, in a Cre-dependent manner. The U6 promoter was selected because the U6 promoter has been widely used for shRNA expression [47,48,49]. In addition, the previous study [47] suggested that the U6 promoter is suitable for Cre-mediated shRNA expression compared with other Pol III promoters, such as the H1 promoter. Human embryonic kidney (HEK) 293T cells were maintained in DMEM (Invitrogen) containing 10% fetal bovine serum, 1% penicillin/streptomycin at 37°C and 5% CO2 in air. The AAV-DJ capsid plasmid (Cell Biolabs, San Diego, CA, USA), pHelper (Cell Biolabs), and AAV-U6-shRNA were co-transfected into HEK293T cells using polyethyleneimine transfection reagent (Sigma-Aldrich). Three days after the transfection at 37°C and 3% CO2 in air, virus vectors were purified by a gradient purification method using iodixanol OptiPrep (Serumwerk Bernburg, Bernburg, Germany), and the titer was quantified using quantitative PCR.

Table 1. DNA sequences of rat Ncor2-targeted shRNA #1 or its scrambled shRNA.

graphic file with name jrd-71-071-t001.jpg

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Effects of ARC Kiss1-dependent Ncor2 knockdown on ARC Kiss1 expression and LH secretion in OVX + high E2 Kiss1-Cre virgin rats and OVX + low E2 Kiss1-Cre lactating rats

AAV-DJ vectors were stereotaxically injected into the ARC of Kiss1-Cre female rats, as described previously [43]. Briefly, AAV vectors carrying U6 promoter-driven Cre-activated Ncor2-targeting shRNA #1 (AAV-U6-[Ncor2-shRNA #1 sense-loxP-stop-loxP-Ncor2-shRNA #1 anti-sense]-CMV-EGFP, 1.16 × 108 viral genomes [vg]/μl) or scrambled-shRNA control (AAV-U6-[Scrambled-shRNA sense-loxP-stop-loxP-Scrambled-shRNA anti-sense]-CMV-EGFP, 0.32 × 108 vg/μl) were bilaterally injected into the anterior and posterior ARC of Kiss1-Cre virgin cyclic female rats (Ncor2-shRNA #1, n = 8; scrambled-shRNA, n = 6) or Kiss1-Cre lactating rats (Ncor2-shRNA #1, n = 8; scrambled-shRNA, n = 4) on day 2 of lactation under anesthesia with a mixture of medetomidine (0.375 mg/kg, ZENOAQ, Koriyama, Japan), midazolam (2 mg/kg, SANDOZ, Tokyo, Japan), and butorphanol (2.5 mg/kg, Meiji Animal Health, Kumamoto, Japan). The stereotaxic coordinates for the injections were 2.8 mm (anterior ARC) and 3.8 mm (posterior ARC) posterior, 10.3 mm ventral to the bregma, and 0.6 mm lateral to the midline, based on the rat brain atlas [38]. Each of the four bilateral injection sites received 1 μl of the vector at a flow rate of 0.25 μl/min over 4 min. After the surgery, the animals were awakened with an injection of atipamezole (0.75 mg/kg, ZENOAQ), a medetomidine antagonist. Three weeks after the AAV injection, Kiss1-Cre virgin female rats treated with Ncor2-shRNA #1 or scrambled-shRNA were subjected to high E2 treatment, as described earlier. OVX + high E2 Kiss1-Cre virgin rats were subjected to blood sampling to determine LH pulses. OVX + low E2 Kiss1-Cre lactating rats treated with AAV on day 2 postpartum were subjected to blood sampling on day 16 postpartum to assess LH pulses. On the day before blood sampling, a silicon cannula (inner diameter 0.5 mm, outer diameter 1.0 mm; Shin-Etsu Polymer, Tokyo, Japan) was inserted into the right atrium via the jugular vein in the AAV-treated Kiss1-Cre virgin and lactating rats. Blood samples (100 μl) were collected from freely moving conscious OVX + high E2 Kiss1-Cre virgin female rats every 6 min for 3 h from 1000 to 1300 h to avoid detecting the afternoon LH surge. Blood samples were similarly collected from OVX + low E2 Kiss1-Cre lactating rats from 1300 to 1600 h at 6-min intervals, as previously described [24]. An equivalent volume of rat red blood cells, obtained from donor rats and diluted with heparinized saline, was replaced through the atrial catheter after each blood collection to maintain a constant hematocrit. Plasma samples were isolated following centrifugation at 4°C.

After blood sampling, the animals were perfused with PBS followed by 4% paraformaldehyde. The brains were collected and sectioned. The sections including the ARC region (from 1.72 to 4.36 mm posterior to the bregma, every fourth section, 13 sections in total) were subjected to ISH for ARC Kiss1 mRNA expression, as previously described [12]. The sections were hybridized with a 1 μg/ml DIG-labeled Kiss1 anti-sense cRNA probe at 60°C overnight. The hybridized probe was detected with an alkaline phosphatase-conjugated anti-DIG antibody (RRID: AB_2734716; 1:1000; Roche Diagnostics) for 3 h at 37°C. The sections were treated with a chromogen solution (338 μg/ml 4-nitroblue tetrazolium chloride and 175 μg/ml 5-bromo-4-chloro-3-indoyl-phosphate) for 3 h at 37°C.

The brain sections were mounted, and the signals were examined using an optical microscope (BX53; Olympus, Tokyo, Japan). The number of Kiss1-expressing cells in the ARC was counted bilaterally in duplicate and averaged. The intensity of Kiss1 mRNA signals in digital photographs of the ARC was quantitatively analyzed using Fiji software (ImageJ, version 2.1.0/1.53c), as previously described [50, 51].

Double staining for Tac3 and EGFP using ISH for Tac3 and immunohistochemistry for EGFP

To evaluate the transfection efficacy of AAV vectors in the ARC kisspeptin neurons, ISH for Tac3 mRNA and immunohistochemistry for EGFP were performed using brain sections from OVX + high E2 Kiss1-Cre virgin rats and OVX + low E2 Kiss1-Cre lactating rats treated with AAV, as previously described [52]. Another series of brain sections, including the ARC region (from 1.72 to 4.36 mm posterior to the bregma, every fourth section, 13 sections in total), were incubated with an anti-EGFP antibody (RRID: AB_300798, 1:1000; Abcam) for two nights at 4°C. The brain sections were then fixed with 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical, Osaka, Japan) for 10 min and rinsed with 0.05 M PBS containing 0.2% glycine. The sections were incubated with 1 µg/ml DIG-labeled Tac3 cRNA probe overnight at 60°C, as described previously [43]. The DIG-labeled Tac3 signal was visualized using the POD-conjugated anti-DIG antibody, TSA Plus Biotin Kit (1:100, Akoya Bioscience, Marlborough, MA, USA), and DyLight 594-conjugated streptavidin (1:500, Thermo Fisher Scientific). The anti-EGFP signal was visualized using Alexa Fluor 488 goat anti-chicken IgY secondary antibody (RRID: AB_2534096, 1:800; Thermo Fisher Scientific) for 2 h at room temperature. Fluorescent images were obtained under a fluorescence microscope with ApoTome2 optical sectioning (Carl Zeiss, Oberkochen, Germany). Tac3- and EGFP-positive cells were counted bilaterally on the photomicrographs.

Radioimmunoassay and LH pulse parameter analysis

Plasma LH concentrations were determined using a double-antibody radioimmunoassay (RIA) with a rat LH-RIA kit provided by the National Hormone and Peptide Program (Harbor-UCLA Medical Center, Torrance, CA, USA). Concentrations were expressed in terms of rat LH-RP-3 (National Institute of Diabetes and Digestive and Kidney Diseases). The least detectable level in the LH assay was 3.9 pg/tube, and the intra- and inter-assay coefficients of variation were 6.18% and 11.26% at 0.625 ng/ml, respectively. LH pulses were identified using the PULSAR computer program [53]. The mean LH concentration and the baseline, frequency, and amplitude of LH pulses were calculated during the 3 h sampling period for each individual and then for the group.

Statistical analysis

Statistical differences in ARC Ncor2 mRNA levels among OVX, OVX + low E2, and OVX + high E2 rats were determined by one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test. Statistical differences in the number of Kiss1- or Tac3-expressing cells and Kiss1/Ncor2- or Tac3/Ncor2-co-expressing cells among OVX, OVX + low E2 lactating, and OVX + low E2 non-lactating rats, as well as Ncor2 mRNA and NCOR2 protein levels between the mHypoA-55 cells treated with Ncor2-siRNA candidates and siRNA-NC, were determined by one-way ANOVA followed by Tukey’s HSD test. Statistical differences in the numbers of Tac3-expressing cells and Tac3/EGFP-double-positive cells, the percentage of Tac3-positive cells co-expressing EGFP, the number of Kiss1-expressing cells, the intensity of Kiss1 mRNA signals, and the LH pulse parameters (the mean LH concentration, as well as the baseline, frequency, and amplitude of LH pulses) between Ncor2-shRNA #1- and scrambled-shRNA-treated OVX + high E2 Kiss1-Cre rats and OVX + low E2 Kiss1-Cre lactating rats were determined using Student’s t-test.

Results

Ncor2 expression in ARC and AVPV kisspeptin neurons in Kiss1-tdTomato heterozygous female rats

RNA-seq analysis of kisspeptin neurons isolated from OVX Kiss1-tdTomato heterozygous rats revealed that Ncor2 and Ncor1 were highly expressed in ARC kisspeptin neurons, whereas other corepressor genes, such as Phb2, Nrip1, and Siah2, were scarcely detected (Figs. 1A, B). The mRNA expression of these genes, including Ncor2 and Ncor1, was barely detectable in the AVPV kisspeptin neurons of OVX + high E2 Kiss1-tdTomato heterozygous rats (Fig. 1B). Quantitative RT-PCR analysis using wild-type female rats showed that ARC Ncor2 mRNA expression levels were comparable among the OVX, OVX + low E2, and OVX + high E2 groups (Figs. 1C, D; P > 0.05).

Fig. 1.

Fig. 1.

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Evaluation of estrogen receptor α (ERα) corepressor candidate gene expression in kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV) and nuclear receptor corepressor 2 gene (Ncor2) expression in ARC and AVPV kisspeptin neurons in female rats. (A) Schematic of the procedure for RNA-seq analysis for ERα corepressors using Kiss1-tdTomato heterozygous female rats. The tdTomato-positive kisspeptin neurons were collected from the ARC in ovariectomized (OVX) rats and from the AVPV in OVX rats treated with proestrous levels of estradiol-17β (OVX + high E2). (B) Gene expression profiles of ERα corepressors in the tdTomato-positive ARC or AVPV Kiss1-tdTomato cells obtained from OVX or OVX + high E2 Kiss1-tdTomato heterozygous female rats. The values were normalized by the reads per kilobase per million mapped reads (RPKM) for each mRNA. (C) Schematic of the procedure for quantitative RT-PCR analysis of ARC Ncor2 mRNA expression in the ARC using OVX, OVX + diestrous levels of E2 (OVX + low E2), and OVX + high E2 wild-type female rats. (D) Relative ARC Ncor2 mRNA expression (normalized to the expression level of Actb mRNA) in OVX, OVX + low E2, and OVX + high E2 female rats. (E) Expression of kisspeptin gene (Kiss1, green) and Ncor2 (magenta) mRNA in the ARC of a representative OVX + low E2 wild-type female rat, as detected by in situ hybridization (ISH). (F) The number of ARC cells expressing Kiss1 alone (green column) or both Kiss1 and Ncor2 (green and magenta striped column). (G) Expression of Kiss1 (green) and Ncor2 (magenta) mRNA in the AVPV of a representative OVX + low E2 wild-type female rat. (H) The number of AVPV cells expressing Kiss1 alone (green column) or both Kiss1 and Ncor2 (green and magenta striped column). The insets indicate representative Kiss1, Ncor2, and Kiss1/Ncor2-co-expressing cells (arrows) at higher magnification. Scale bars, 100 μm. Values expressed are mean ± SEM. The number in each column indicates the number of animals used.

Double ISH for Ncor2 and Kiss1 showed that Ncor2 mRNA was expressed in a majority of ARC Kiss1-expressing cells of OVX + low E2 rats (Fig. 1E). Quantitative analysis revealed that 82.6 ± 1.0% of ARC Kiss1-expressing cells exhibited Ncor2 mRNA in OVX + low E2 wild-type rats (Fig. 1F). Ncor2 mRNA expression was also found in the AVPV Kiss1-expressing cells of OVX + low E2 rats (Fig. 1G). Quantitative analysis revealed that 43.0 ± 1.3% of AVPV Kiss1-expressing cells exhibited Ncor2 mRNA in OVX + low E2 wild-type rats (Fig. 1H). Since the proportion of kisspeptin neurons expressing Ncor2 was much higher in ARC kisspeptin neurons than in AVPV kisspeptin neurons, the role of NCOR2 in the estrogen-dependent regulation of ARC Kiss1 expression and GnRH/LH pulse generation in female rats was further investigated.

Ncor2 expression in ARC kisspeptin neurons during late lactation determined by double ISH

The expression of Ncor2 in ARC kisspeptin neurons was analyzed in OVX + low E2 non-lactating rats, OVX + low E2 lactating rats, and OVX lactating rats during late lactation (Fig. 2A). Many ARC Kiss1-expressing cells were found in OVX lactating and OVX + low E2 non-lactating rats, whereas OVX + low E2 lactating rats showed fewer ARC Kiss1-expressing cells compared to the other groups (Fig. 2B). Ncor2 mRNA was observed in the majority of Kiss1-expressing cells in all three groups, and the percentages of Kiss1/Ncor2-co-expressing cells among the total ARC Kiss1-expressing cells were 89.0 ± 2.5% in OVX + low E2 non-lactating rats, 80.2 ± 1.9% in OVX + low E2 lactating rats, and 92.1 ± 1.6% in OVX lactating rats (Fig. 2C).

Fig. 2.

Fig. 2.

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Double ISH for Kiss1 and Ncor2 or neurokinin B gene (Tac3) and Ncor2 mRNA expression in the ARC in OVX + low E2 non-lactating, OVX + low E2 lactating, and OVX lactating wild-type rats. (A) Schematic of the experimental schedule to investigate ARC Kiss1/Tac3 and Ncor2 mRNA expression in non-lactating and lactating rats. The day of delivery was set as day 0 postpartum. On day 1 postpartum, the number of pups was adjusted to eight for lactating rats, and all pups were deprived for non-lactating rats. On day 2 postpartum, non-lactating and lactating rats were OVX and treated with low E2 implantation, while some OVX lactating rats were kept without E2 treatment. Brain samples were collected on day 16 postpartum. (B) Expression of Kiss1 (green) and Ncor2 (magenta) mRNA in the ARC of representative OVX + low E2 non-lactating, OVX + low E2 lactating, and OVX lactating rats. The insets indicate representative Kiss1, Ncor2, and Kiss1/Ncor2-co-expressing cells (arrows) at higher magnification. (C) The number of Kiss1-expressing cells (green column) and the number of Kiss1/Ncor2-co-expressing cells (green and magenta striped column) in OVX + low E2 lactating rats were significantly lower than those in OVX + low E2 non-lactating and OVX lactating rats. Additionally, the number of Kiss1-expressing cells in OVX lactating rats was significantly lower than those in OVX + low E2 non-lactating rats. Values with different letters were significantly different from each other (P < 0.05, one-way ANOVA followed by Tukey’s HSD test). (D) Expression of Tac3 (green) and Ncor2 (magenta) mRNA in the ARC of representative OVX + low E2 non-lactating, OVX + low E2 lactating, and OVX lactating rats. The insets indicate representative Tac3, Ncor2, and Tac3/Ncor2-co-expressing cells (arrows) at higher magnification. (E) The number of ARC cells expressing Tac3 alone (green column) or both Tac3 and Ncor2 (green and magenta striped column) in OVX + low E2 non-lactating, OVX + low E2 lactating, and OVX lactating rats were comparable between groups. Scale bars, 100 μm. Values expressed are mean ± SEM. The number in each column indicates the number of animals used.

Quantitative analysis revealed that the number of ARC Kiss1-expressing cells was significantly lower in OVX + low E2 lactating rats compared to OVX + low E2 non-lactating (P < 0.0001) and OVX lactating rats (P < 0.0001). Additionally, the number of ARC Kiss1-expressing cells was significantly lower in OVX lactating rats compared to OVX + low E2 non-lactating rats (P = 0.0272). The number of ARC Kiss1/Ncor2-co-expressing cells was also significantly lower in OVX + low E2 lactating rats compared to OVX + low E2 non-lactating (P < 0.0001) and OVX lactating rats (P < 0.0001).

Many ARC Tac3 (an indicator of ARC kisspeptin neurons)-expressing cells were observed in all three groups (Fig. 2D). The numbers of ARC Tac3-expressing cells and Tac3/Ncor2-co-expressing cells were comparable between OVX + low E2 non-lactating, OVX + low E2 lactating, and OVX lactating rats (Fig. 2E; P > 0.05). The percentages of Tac3/Ncor2-co-expressing cells among the total ARC Tac3-expressing cells were 87.8 ± 0.6% in OVX + low E2 non-lactating rats, 90.3 ± 1.0% in OVX + low E2 lactating rats, and 88.4 ± 2.0% in OVX lactating rats (Fig. 2E).

Validation of Ncor2 mRNA and protein repression by Ncor2 silencing in mHypoA-55 cell line

Four Ncor2-targeting siRNA candidates (siRNAs #1–#4) were used for in vitro evaluation of their silencing effects (Fig. 3A). Quantitative RT-PCR analysis revealed that Ncor2-targeting siRNAs #1 and #2 significantly reduced Ncor2 mRNA levels in the mHypoA-55 cell line at 24 h (P = 0.0128 and P = 0.0188, respectively) and 48 h (P = 0.0211 and P = 0.0228, respectively) after transfection compared to the untreated group (Fig. 3B). Ncor2-targeting siRNAs #3 and #4 significantly reduced Ncor2 mRNA levels at 24 h (P = 0.0122 and P = 0.0228, respectively); however, the mRNA levels partly recovered 48 h after transfection (Fig. 3B). In addition, western blot analysis demonstrated that Ncor2-targeted siRNA #1, but not siRNA #2, significantly reduced NCOR2 protein levels in mHypoA-55 cells 48 h after transfection (Fig. 3C; P = 0.0377).

Fig. 3.

Fig. 3.

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In vitro validation of Ncor2-siRNA candidates to repress Ncor2 mRNA expression and NCOR2 protein expression in the mHypoA-55 cell line. (A) Schematic of the procedure for evaluating Ncor2-siRNA in the mHypoA-55 cell line. mHypoA-55 cells were transfected with one of four Ncor2 siRNA candidates (siRNA #1–#4) or The MISSION siRNA Universal Negative Control #1 (siRNA-NC). Following transfection, the cells were cultured for an additional 24 or 48 h and treated with E2 for 2 h prior to collection. (B) Ncor2 mRNA expression was analyzed by quantitative RT-PCR 24 or 48 h after Ncor2-siRNA treatment, with expression levels normalized to Actb mRNA levels. Values with different letters were significantly different from each other (P < 0.05, one-way ANOVA followed by Tukey’s HSD test). (C) NCOR2 and β-actin protein expression levels were analyzed by western blotting. NCOR2 protein levels were normalized to β-actin protein levels. Values with different letters were significantly different from each other (P < 0.05, one-way ANOVA followed by Tukey’s HSD test). Ncor2-targeting siRNA #1 significantly reduced both Ncor2 mRNA and NCOR2 protein levels in the mHypoA-55 cell line compared to the untreated group. The number in each column indicates the number of cell culture replicates used.

Ncor2 knockdown partly blocked estrogen-induced ARC Kiss1 repression in female rats

We first investigated the effects of ARC Kiss1-dependent Ncor2 knockdown using an AAV vector carrying Ncor2-shRNA #1, corresponding to siRNA #1, on ARC Kiss1 expression in OVX + high E2 Kiss1-Cre rats (Fig. 4A). Representative images of Tac3/EGFP-double-positive cells in the anterior and posterior ARC of OVX + high E2 Kiss1-Cre virgin rats transfected with Ncor2-targeting shRNA #1 or scrambled-shRNA are shown in Fig. 4B. Quantitative analysis revealed that 28.1 ± 3.6% and 23.3 ± 2.0% of ARC Tac3-expressing cells exhibited EGFP immunoreactivity in Ncor2-shRNA #1- or scrambled-shRNA-treated OVX + high E2 Kiss1-Cre rats, respectively. No significant differences were observed in the numbers of Tac3-expressing cells (P = 0.8627) and Tac3/EGFP-double-positive cells (P = 0.4081), and the percentages of Tac3-positive cells co-expressing EGFP (P = 0.3712) between the Ncor2-shRNA #1- and scrambled-shRNA-treated groups in OVX + high E2 Kiss1-Cre rats (Fig. 4C). Representative images of Kiss1-expressing cells in the anterior and posterior ARC of OVX + high E2 Kiss1-Cre virgin rats transfected with Ncor2-targeting shRNA #1 or scrambled-shRNA are shown in Fig. 4D. Quantitative analysis revealed that the number of ARC Kiss1-expressing cells was significantly higher in Kiss1-Cre rats transfected with Ncor2-shRNA #1 compared to Kiss1-Cre control rats transfected with scrambled-shRNA (Fig. 4E; P = 0.0276). Moreover, the intensity of Kiss1 mRNA signals was significantly higher in the Ncor2-shRNA #1 group than in the control group (Fig. 4E; P = 0.0235).

Fig. 4.

Fig. 4.

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Effects of Ncor2-shRNA administration into the ARC on Kiss1 repression in the ARC of OVX + high E2 Kiss1-Cre virgin rats. (A) Schematic of the procedure for Ncor2 knockdown in ARC Kiss1-expressing cells in OVX + high E2 Kiss1-Cre virgin rats. An adeno-associated virus (AAV) vector carrying Cre-dependent Ncor2-shRNA #1, corresponding to siRNA #1, or scrambled-shRNA (control) with a Cre-independent CMV promoter-driven EGFP reporter was bilaterally administered into the anterior and posterior ARC of Kiss1-Cre virgin rats. Animals were OVX three weeks after AAV administration and treated with low E2 for five days and high E2 for two days. Blood and brain samples were collected under the high E2 condition. (B) Tac3 (a marker for ARC kisspeptin neurons)-expressing cells detected by ISH (magenta) and EGFP protein detected by immunohistochemistry (green) in the anterior and posterior ARC of representative OVX + high E2 Kiss1-Cre virgin rats treated with Ncor2-shRNA #1 or scrambled-shRNA. (C) The numbers of ARC Tac3-expressing cells (magenta column) and Tac3/EGFP-double-positive cells (magenta and green striped column), and the percentages of Tac3-positive cells co-expressing EGFP in the Ncor2-shRNA- or scrambled-shRNA-treated groups. The numbers of Tac3-expressing cells and Tac3/EGFP-double-positive cells, and the percentages were comparable between the Ncor2-shRNA #1- and scrambled-shRNA-treated groups. (D) Kiss1-expressing cells detected by ISH in the ARC of representative OVX + high E2 Kiss1-Cre virgin rats treated with Ncor2-shRNA #1 or scrambled-shRNA. (E) The number of ARC Kiss1-expressing cells in OVX + high E2 Kiss1-Cre virgin rats treated with Ncor2-shRNA #1 was significantly higher than that in scrambled-shRNA-treated controls (* P < 0.05, Student’s t-test). The intensity of ARC Kiss1 mRNA signals in OVX + high E2 Kiss1-Cre virgin rats treated with Ncor2-shRNA #1 was significantly higher than that in scrambled-shRNA-treated controls (* P < 0.05, Student’s t-test). The insets indicate representative Tac3/EGFP-double-positive cells and Kiss1-expressing cells (arrows) at higher magnification. Scale bars, 100 μm. The number in each column indicates the number of animals used.

Next, we examined the effect of ARC Kiss1-dependent Ncor2 knockdown on ARC Kiss1 expression in OVX + low E2 Kiss1-Cre lactating rats (Fig. 5A). Representative images of Tac3/EGFP-double-positive cells in the anterior and posterior ARC of OVX + low E2 Kiss1-Cre lactating rats treated with Ncor2-shRNA #1 or scrambled-shRNA are shown in Fig. 5B. Quantitative analysis revealed that 35.4 ± 3.5% and 27.0 ± 5.3% of Tac3-expressing cells exhibited EGFP immunoreactivity in Ncor2-shRNA- or scrambled-shRNA-treated OVX + low E2 Kiss1-Cre lactating rats, respectively. No significant differences were observed in the numbers of Tac3-expressing cells (P = 0.8890) and Tac3/EGFP-double-positive cells (P = 0.2261), and the percentages of Tac3-positive cells co-expressing EGFP (P = 0.2242) between the Ncor2-shRNA #1- and scrambled-shRNA-treated groups in OVX + low E2 Kiss1-Cre lactating rats (Fig. 5C). A few Kiss1-expressing cells were found in the ARC of OVX + low E2 Kiss1-Cre lactating rats transfected with either Ncor2-shRNA #1 or scrambled-shRNA (Fig. 5D). The number of Kiss1-expressing cells (P = 0.3025) and the intensity of Kiss1 mRNA signals (P = 0.3221) tended to be higher in OVX + low E2 Kiss1-Cre lactating rats transfected with Ncor2-shRNA #1 compared to scrambled-shRNA-treated rats (Fig. 5E).

Fig. 5.

Fig. 5.

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The effects of Ncor2-shRNA administration into the ARC on Kiss1 mRNA expression in OVX + low E2 Kiss1-Cre lactating rats. (A) Schematic of the procedure for Ncor2 knockdown in ARC Kiss1-expressing cells in OVX + low E2 Kiss1-Cre lactating rats. The day of delivery was designated as day 0 postpartum, and the number of pups was adjusted to eight on day 1 postpartum. On day 2 postpartum, Kiss1-Cre lactating rats were OVX and implanted with low E2, and AAV carrying Cre-dependent Ncor2-shRNA #1 or scrambled-shRNA and a Cre-independent CMV promoter-driven EGFP reporter was bilaterally administered into the anterior and posterior ARC. Blood and brain samples were collected on day 16 postpartum. (B) Tac3-expressing cells (magenta) and EGFP-immunopositive cells (green) in the anterior and posterior ARC of representative OVX + low E2 Kiss1-Cre lactating rats treated with Ncor2-shRNA #1 or scrambled-shRNA. (C) The numbers of Tac3-expressing cells (magenta column) and Tac3/EGFP-double-positive cells (magenta and green striped column), and the percentages of Tac3-positive cells co-expressing EGFP in the Ncor2-shRNA- or scrambled-shRNA-treated groups. The numbers of Tac3-expressing cells and Tac3/EGFP-double-positive cells, and the percentages were comparable between the Ncor2-shRNA #1- and scrambled-shRNA-treated groups. (D) Kiss1-expressing cells in the ARC of representative OVX + low E2 Kiss1-Cre lactating rats treated with Ncor2-shRNA #1 or scrambled-shRNA. (E) The number of ARC Kiss1-expressing cells in OVX + low E2 Kiss1-Cre lactating rats was comparable between the Ncor2-shRNA #1- and scrambled-shRNA-treated groups. The intensity of ARC Kiss1 mRNA signals in OVX + low E2 Kiss1-Cre lactating rats was comparable between the Ncor2-shRNA #1- and scrambled-shRNA-treated groups. The insets indicate representative Tac3/EGFP-double-positive cells and Kiss1-expressing cells (arrows) at higher magnification. Scale bars, 100 μm. The number in each column indicates the number of animals used.

Ncor2 knockdown failed to block estrogen-induced LH pulse suppression in female rats

Representative plasma LH profiles of OVX + high E2 Kiss1-Cre rats treated with Ncor2-shRNA #1 or scrambled-shRNA are shown in Fig. 6A. Some rats in both groups exhibited suppressed LH pulses, while others displayed clear LH pulses. The mean plasma LH concentrations were comparable between OVX + high E2 Kiss1-Cre rats treated with Ncor2-shRNA #1 and those treated with scrambled-shRNA (P = 0.7343). Similarly, no significant differences were observed in the baseline (P = 0.8877), frequency (P = 0.7130), or amplitude (P = 0.8573) of LH pulses between the two groups (Fig. 6B). Figure 6C presents plasma LH profiles from representative OVX + low E2 Kiss1-Cre lactating rats treated with Ncor2-shRNA #1 or scrambled-shRNA. The mean LH concentrations (P = 0.1626), and the baseline (P = 0.1387) and frequency (P = 0.1316) of LH pulses tended to be higher in Ncor2-shRNA #1-transfected OVX + low E2 lactating rats compared to scrambled-shRNA-treated lactating rats (Fig. 6D).

Fig. 6.

Fig. 6.

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Administration of Ncor2-shRNA into the ARC failed to affect pulsatile luteinizing hormone (LH) release in both OVX + high E2 Kiss1-Cre virgin rats and OVX + low E2 Kiss1-Cre lactating rats. (A) Plasma LH profiles in OVX + high E2 Kiss1-Cre rats administered with AAV carrying Ncor2-shRNA #1 or scrambled-shRNA (control) into the bilateral anterior and posterior ARC as described in Figs. 4A and 5A. Arrowheads indicate the peaks of LH pulses identified by the PULSAR computer program. (B) The mean plasma LH concentrations, as well as the baseline, frequency, and amplitude of LH pulses, were comparable between the groups (P > 0.7, Student’s t-test). (C) Plasma LH profiles in OVX + low E2 Kiss1-Cre lactating rats injected with Ncor2-shRNA #1 or scrambled-shRNA into the ARC. (D) The mean LH concentrations (P = 0.1626), as well as the baseline (P = 0.1387) and frequency (P = 0.1316) of LH pulses, tended to be higher in the Ncor2-shRNA #1-transfected OVX + low E2 lactating rats than in scrambled-shRNA-treated lactating rats (Student’s t-test). The numbers in each column indicate the number of animals used.

Discussion

This study demonstrated that NCOR2, an ERα corepressor, partly mediates the repression of ARC Kiss1 expression induced by proestrous levels of estrogen in virgin female rats. This is because the majority (> 80%) of ARC kisspeptin neurons expressed Ncor2, and Kiss1-dependent Ncor2 knockdown significantly increased both the number of ARC Kiss1-expressing cells and the intensity of Kiss1 mRNA signals in OVX + high E2 Kiss1-Cre virgin rats. NCOR2, which functions as an adaptor to recruit HDAC, can bind to ERα in an estrogen-dependent manner to repress target genes by inducing histone deacetylation [54]. This notion is consistent with previous studies showing that high (proestrous) levels of E2 profoundly repressed ARC Kiss1 mRNA expression in OVX rats and mice [3, 12, 20, 31, 55] and significantly decreased histone acetylation at the Kiss1 promoter region in the ARC of mice [31]. Therefore, the current ARC Kiss1-dependent Ncor2 silencing likely prevented estrogen-induced histone deacetylation at the ARC Kiss1 promoter, thereby partially blocking high E2-induced Kiss1 repression in ARC kisspeptin neurons in female rats.

This study, however, failed to evaluate the role of NCOR2 in low E2-induced repression of ARC Kiss1 expression in lactating rats because Kiss1-dependent Ncor2 knockdown failed to fully restore the number of ARC Kiss1-expressing cells or the intensity of Kiss1 mRNA signals in OVX + low E2 Kiss1-Cre lactating rats during late lactation. Notably, our previous study demonstrated that central antagonism of type-2 somatostatin receptor (SSTR2) signaling significantly increased the number of ARC Kiss1-expressing cells in OVX lactating rats treated with low E2 during late lactation [56]. This suggests that inhibitory inputs, such as somatostatin-SSTR2 signaling, may partly contribute to the low E2-induced repression of ARC Kiss1 expression in lactating rats. Moreover, conditional knockout of the prolactin receptor (PRLR) in neurons expressing calcium/calmodulin-dependent protein kinase IIα, which is highly expressed in forebrain neurons, increased kisspeptin immunoreactivity in the ARC of lactating mice [57]. This implies that PRLR-mediated inhibitory regulation may contribute to repressing kisspeptin expression in mice during lactation. Alternatively, other intracellular ERα-mediated repressive cofactors, such as DAX1 and SP3, may play a role in the lactational repression of ARC Kiss1 expression, because DAX1 was reported to repress in vivoKiss1 expression in the mouse hypothalamus [58], and SP3 was shown to repress in vitroKiss1 expression in HEK293T cells [59]. Further studies are necessary to clarify the roles of NCOR2 and/or other ERα corepressors in low E2-induced ARC Kiss1 repression during lactation.

The present study demonstrated that Kiss1-dependent Ncor2 knockdown failed to restore LH pulses in OVX + high E2 Kiss1-Cre rats, despite a significant increase in the number of ARC Kiss1-expressing cells and the intensity of Kiss1 mRNA signals in Ncor2-shRNA #1-treated OVX + high E2 virgin rats compared to scrambled-shRNA-treated OVX + high E2 Kiss1-Cre control rats. This inconsistency between ARC Kiss1 expression and LH pulses may partly result from inefficient Ncor2 knockdown in ARC kisspeptin neurons. Alternatively, NCOR2 may be involved in the repression of high E2-induced ARC Kiss1 expression, but not in kisspeptin release. Furthermore, the current Kiss1-dependent Ncor2 knockdown failed to significantly increase the number of ARC Kiss1-expressing cells, the intensity of Kiss1 mRNA signals, or LH pulses in OVX + low E2 Kiss1-Cre lactating rats. Previous studies revealed that low E2 treatment enhanced paraventricular nucleus (PVN) dynorphin A (Dyn) expression in OVX virgin rats, and PVN Dyn neurons mediated lactational or glucoprivic LH pulse suppression in female rats [35, 60]. Thus, the repression of Kiss1 mRNA expression and kisspeptin release may be regulated by distinct mechanisms under negative feedback levels of estrogen in female rats.

In conclusion, this study demonstrated that NCOR2 in ARC kisspeptin neurons partly mediates the repression of ARC Kiss1 expression in the presence of proestrous levels of estrogen in female rats. On the other hand, the role of NCOR2 in low E2-induced ARC Kiss1 repression in lactating rats could not be evaluated. Further studies are required to investigate the role of NCOR2 and/or other intracellular factors involved in the repression of ARC Kiss1 expression in lactating rats under diestrous levels of estrogen.

Conflict of interests

The authors declare that they have no competing interests.

Acknowledgments

The authors thank the National Hormone and Peptide Program for providing rat LH-RIA kits and Drs. G.R. Merriam and K.W. Wachter for the PULSAR computer program. Radioimmunoassays were performed at the Radioisotope Research Center, Nagoya University, Japan. This work was supported by the Japan Society for the Promotion of Science KAKENHI grant numbers JP21H05031, JP21K19186 (to H. Tsukamura), JP23H02362, JP23K27055 (to N.I.), and JP20H03127, JP22K19245, JP24K01905 (to Y.U.); the Graduate Program of Transformative Chem-Bio Research in Nagoya University, supported by MEXT (WISE Program) (to H. Tsuchida and M.N.); and “Nagoya University Interdisciplinary Frontier Fellowship” supported by Nagoya University and the Japan Science and Technology Agency, the establishment of university fellowships towards the creation of science technology innovation, grant number JPMJFS2120 (to M.T.).

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