Pubertal Escape From Estradiol Negative Feedback in Ewe Lambs Is Not Accounted for by Decreased ESR1 mRNA or Protein in Kisspeptin Neurons

Pubertal Escape From Estradiol Negative Feedback in Ewe Lambs Is Not Accounted for by Decreased... Abstract In this study, we investigated whether decreased sensitivity to estradiol negative feedback is associated with reduced estrogen receptor α (ESR1) expression in kisspeptin neurons as ewe lambs approach puberty. Lambs were ovariectomized and received no implant (OVX) or an implant containing estradiol (OVX+E). In the middle arcuate nucleus (mARC), ESR1 messenger RNA (mRNA) was greater in OVX than OVX+E lambs but did not differ elsewhere. Post hoc analysis of luteinizing hormone (LH) secretion from OVX+E lambs revealed three patterns of LH pulsatility: low [1 to 2 pulses per 12 hours; low frequency (LF), n = 3], moderate [6 to 7 pulses per 12 hours; moderate frequency (MF), n = 6], and high [>10 pulses per 12 hours; high frequency (HF), n = 5]. The percentage of kisspeptin neurons containing ESR1 mRNA in the preoptic area did not differ among HF, MF, or LF groups. However, the percentage of kisspeptin neurons containing ESR1 mRNA in the mARC was greater in HF (57%) than in MF (36%) or LF (27%) lambs and did not differ from OVX (50%) lambs. A higher percentage of kisspeptin neurons contained ESR1 protein in all regions of the arcuate nucleus (ARC) in OVX compared with OVX+E lambs. There were no differences in ESR1 protein among the HF, MF, or LF groups in the preoptic area or ARC. Contrary to our hypothesis, increases in LH pulsatility were associated with enhanced ESR1 mRNA abundance in kisspeptin neurons in the ARC, and absence of estradiol increased the percentage of kisspeptin neurons containing ESR1 protein in the ARC. Therefore, changes in the expression of ESR1, particularly in kisspeptin neurons in the ARC, do not explain the pubertal escape from estradiol negative feedback in ewe lambs. Puberty is a well-characterized reproductive event that has been investigated extensively in female mammals. As female sheep approach reproductive maturation, the frequency of luteinizing hormone (LH) pulses increases (1–3). It is known that activation of GnRH neurons in the preoptic area and hypothalamus, as well as secretion of GnRH into the hypothalamic–hypophyseal portal vasculature, is critical for the initiation of the pubertal pattern of LH release (4, 5). However, the neuroendocrine mechanisms involved in controlling the onset of high-frequency GnRH/LH release are still unclear. Whereas a decline in estradiol negative feedback has not been definitively shown to play a major role in pubertal onset across all species, including nonhuman primates (6) and rats (7), a decline in estradiol negative feedback is absolutely essential for the increase in GnRH and LH pulse frequency associated with the initiation of puberty in sheep (2, 8). However, GnRH neurons do not contain estrogen receptor α (ESR1), which is thought to be the major estrogen receptor mediating estradiol’s regulation of GnRH secretion (9). Another estrogen receptor, estrogen receptor β, is expressed in a subpopulation of GnRH neurons in sheep (10), but it does not appear to be important for the control of reproductive neuroendocrine processes (9). Therefore, it is hypothesized that afferent neurons containing ESR1 in the hypothalamus mediate the feedback effects of estradiol on GnRH neurons. Kisspeptin neurons contain ESR1 (11), and kisspeptin stimulates GnRH secretion (12). The absence of Esr1 in kisspeptin neurons has also been observed to prematurely activate pulsatile secretion of LH, increase Kiss1 messenger RNA (mRNA) abundance, and advance the day of vaginal opening in female mice (13, 14). Therefore, elucidating the control of ESR1 expression in kisspeptin neurons could reveal important information regarding estradiol’s role in controlling GnRH secretion and reproductive cyclicity. The present study investigated the hypothesis that decreased sensitivity to estradiol negative feedback and subsequent increases in LH pulsatility in peripubertal female sheep are associated with reduced ESR1 mRNA abundance and protein content in kisspeptin neurons. Materials and Methods Animal experiments were conducted at the Texas A&M University Nutrition and Physiology Center located in the O.D. Butler Animal Science Teaching and Research Complex in College Station, Texas. All procedures used in these studies were approved by the Institutional Agricultural Animal Care and Use Committee of the Texas A&M University and followed National Institutes of Health guidelines for use of animals in research. Animals and experimental procedures Spring-born ewe lambs (n = 21) were ovariectomized at ∼24 weeks of age. Lambs were housed in individual pens and fed ad libitum a commercial diet formulated to meet National Research Council recommendations for growing lambs. At the time of ovariectomy, ewe lambs were selected randomly to receive either no implant (OVX; n = 7) or a subcutaneous implant containing crystalline estradiol (Sigma-Aldrich, St. Louis, MO) (OVX+E; n = 14). The estradiol implants were designed to produce circulating concentrations of estradiol of ∼1 to 2 pg/mL and successfully maintain estradiol negative feedback on gonadotropin release in ovariectomized ewe lambs (8), as reported previously from this laboratory (15). At 30 weeks of age, a time when ewes are approaching pubertal transition, a catheter (16-gauge by 3 inches in polyurethane; Jorgensen Laboratories, Loveland, CO) was inserted into a jugular vein 24 hours before initial blood sampling. The next day, lambs were restrained loosely in their home pen with a halter and blood samples (5 mL) were collected remotely every 10 minutes for 12 hours using an extension connected to the jugular catheter. Lambs were acclimated to experimental conditions of blood collection for 5 days before blood sampling and were housed adjacent to other sheep at all times. Immediately after collection, blood samples were placed in tubes containing 50 µL of a solution of heparin (3000 U/mL) and 5% EDTA and mixed gently. Samples were placed immediately on ice and centrifuged at 2200 × g for 20 minutes at 4°C within 2 hours of collection. Plasma was collected and stored at −20°C until processing to determine concentrations of LH. On the day after intensive collection of blood samples, ewe lambs were euthanized with an overdose of pentobarbital (Beuthanasia-D Special; Schering-Plough, Union, NJ), and heads were perfused with a solution containing 4% paraformaldehyde. Brains were dissected from the cranium and a block of tissue containing the septum, preoptic area (POA), and hypothalamus was collected and placed in 4% paraformaldehyde at 4°C for 48 hours, with paraformaldehyde solution replaced after 24 hours. After paraformaldehyde incubation, tissue blocks were placed in a 0.1 M phosphate-buffered solution containing 30% sucrose at 4°C for at least 7 days. Tissue processing Tissue blocks were sectioned in coronal sections of 50 µm using a freezing microtome (Microm HM430; Microm International, Walldorf, Germany). Sections were then stored at −20°C in a cryopreservative solution until processed for double-label in situ hybridization and immunocytochemistry for ESR1 and kisspeptin or double-label immunocytochemistry for ESR1 and kisspeptin. Double-label in situ hybridization and immunocytochemistry To detect ESR1 mRNA, sense and antisense radiolabeled complementary RNA probes were generated by in vitro transcription of a DNA template containing a 311-bp sequence of ovine ESR1 complementary DNA (cDNA) linked to RNA polymerase promoters. The template was produced by polymerase chain reaction (PCR) from the linearized poER8 plasmid containing a partial ovine ESR1 cDNA provided graciously by Dr. Nancy Ing (Texas A&M University; GenBank accession number U30299.1) (16), following procedures described previously (17). The initial PCR used the primers 5′-CGAGCGGCTATGCGGTG-3′ and 5′-GGCCTGACAGCTCTTCCTTC-3′ to amplify an ESR1-specific sequence. A second reaction was performed using the product of the first reaction as a template and primers tagged with the T3 and T7 RNA polymerase promoter sequences. Primers used in the second reaction were 5′-AATTAACCCTCACTAAAGGGCGAGCGGCTATGCGGTG-3′ (T3 promoter in bold type) and 5′-TAATACGACTCACTATAGGGAGGCCTGACAGCTCTTCCTTC-3′ (T7 promoter in bold type). The PCR product was sequenced and the sequence obtained was aligned to the available Ovis aries genomic database using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The cDNA obtained was 100% identical to the ovine ESR1 cDNA. The in vitro transcription reaction for synthesis of 35S-labeled probes followed procedures described previously (15). Free-floating sections were washed in 0.1 M phosphate buffer 10 times for 6 minutes each. The sections were then placed in a 1% NaBH4 solution for 15 minutes. Sections were washed in 0.1 M phosphate buffer 10 times for 6 minutes each and then washed in 0.1 M triethanolamine twice for 10 minutes. Next, sections were incubated in a 0.25% acetic anhydride solution for 10 minutes before being washed in 2× saline sodium citrate (SSC) three times for 10 minutes. Sections were then hybridized with sense or antisense radiolabeled complementary RNA probes for ESR1 overnight at 55°C. Before hybridization, probes were diluted in hybridization buffer (50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl, 5 mM EDTA, 10 mM NaPO4, Denhardt solution, 10% dextran sulfate, 0.5 mg/ml yeast transfer RNA, and 100 mM dithiothreitol) and denatured at 70°C for 10 minutes. After overnight incubation, the sections were briefly washed four times in 4× SSC and then treated with RNase in a buffer containing Tris-Cl, EDTA, and NaCl for 30 minutes at 37°C. The sections were then incubated in a 1× Tris-Cl, EDTA, and NaCl buffer for 30 minutes at 37°C followed by washes in 2× SSC for 30 minutes at 55°C and 0.2× SSC for 30 minutes at 55°C. After completion of the in situ hybridization procedure, tissue sections were immediately processed for immunodetection of kisspeptin. Sections were washed in phosphate-buffered saline (PBS) four times for 5 minutes and then incubated in PBS containing 1% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity. After further washing, sections were incubated in a solution of PBS containing 0.4% Triton X-100 (PBSTX) and 4% normal goat serum (NGS) for at least 1 hour. Sections were then incubated in a solution containing rabbit anti-kisspeptin antiserum [AC no. 564; 1:75,000; Research Resource Identifier (RRID): AB_2622231; provided by Dr. Alain Caraty, Institut National de la Recherche Agronomique, Nouzilly, France] in PBSTX and 4% NGS for 16 hours. After incubation with the primary antibody, sections were washed and incubated in a solution containing biotinylated goat anti-rabbit IgG (1:400; Vector Laboratories, Burlingame, CA), PBSTX, and 4% NGS for 1 hour. The sections were washed and incubated in a solution containing streptavidin–horseradish peroxidase conjugate (Vectastain Elite ABC; 1:600; Vector Laboratories) for 1 hour. Sections were then washed and incubated in a solution containing 3,3′-diaminobenzidine (DAB; 0.2 mg/mL) with hydrogen peroxide (0.012%; Sigma-Aldrich) in PBS for 10 minutes. The sections were washed again, mounted on slides, and dried at 37°C. Slides were dipped in photographic NBT2 emulsion (Kodak Company, Rochester, NY), dried, and exposed in the dark for 35 days at 4°C. Slides were developed in D-19 developer and sections were counterstained with cresyl violet, dehydrated, and covered with glass slips using DPX (VWR International, Radnor, PA). Double-label immunocytochemistry Similar to the above immunocytochemical protocol, sections were washed in PBS four times for 5 minutes and then incubated in PBS containing 1% hydrogen peroxide for 10 minutes. After further washing, the sections were incubated in a solution of PBS containing 0.4% PBSTX and 4% NGS for at least 1 hour. The sections were then incubated in a solution containing mouse anti-ESR1 antiserum (catalog no. M7047; clone 1D5; RRID: AB_2101946; 1:500; DAKO, Glostrup, Denmark) in PBSTX and 4% NGS for 16 hours. After incubation with the primary antibody, sections were washed and then incubated in a solution containing biotinylated goat anti-mouse IgG (1:400; Vector Laboratories), PBSTX, and 4% NGS for 1 hour. The sections were washed and incubated in a solution containing streptavidin–horseradish peroxidase conjugate (Vectastain Elite ABC; 1:600; Vector Laboratories) for 1 hour. Sections were then washed and incubated in a solution containing DAB (0.2 mg/mL), nickel sulfate (2%; Sigma-Aldrich), and hydrogen peroxide (0.012%; Sigma-Aldrich) in PBS for 10 minutes. The sections were washed and incubated in PBS containing 1% hydrogen peroxide for 10 minutes. After further washing, the sections were incubated in PBSTX and 4% NGS for at least 1 hour. The sections were then incubated in a solution containing rabbit anti-kisspeptin antiserum (catalog no. AB9754; RRID: AB_229652; 1:2000; Millipore, Billerica, MA) in PBSTX and 4% NGS for 16 hours. After incubation with the primary antibody, the sections were washed and then incubated in a solution containing biotinylated goat anti-rabbit IgG (1:400; Vector Laboratories), PBSTX, and 4% NGS for 1 hour. The sections were washed and incubated in a solution containing streptavidin–horseradish peroxidase conjugate (Vectastain Elite ABC; 1:600; Vector Laboratories) for 1 hour. Sections were then washed and incubated in a solution containing DAB (0.2 mg/mL) with hydrogen peroxide (0.012%; Sigma-Aldrich) in PBS for 10 minutes. The sections were washed, mounted on slides, and dried at 37°C. Slides were dehydrated and covered with glass slips using DPX (VWR International). ESR1 mRNA abundance in the POA and hypothalamus Processed slides were coded and tissue sections were analyzed by an observer who was unaware of each animal’s experimental group. Overall ESR1 mRNA abundance in the POA and hypothalamus was determined using a dark- and bright-field microscope (Nikon 80i Eclipse; Nikon, Melville, NY). The anatomical location and distribution of ESR1 mRNA was investigated in the POA, periventricular nucleus (PeV), supraoptic nucleus (SON), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), ventrolateral–ventromedial hypothalamus (VL-VMH), and arcuate nucleus (ARC). For each region examined, four sections within the POA [including two sections at the level of the organum vasculosum of the lamina terminalis (OVLT)], three through the PeV, four through the SON, four through the PVN, two through the rostral ARC (rARC), four through the middle ARC (mARC), two through the caudal ARC (cARC), three through the VMH, and two through the VL-VMH were selected. Sections were selected to represent comparable levels within each area examined. Bright-field images of each region were captured using a ×40 objective and a digital camera (DS-Qi1; Nikon) attached to the microscope. In the SON, VMH, and VL-VMH, three images were collected per section analyzed. In the POA, PeV, PVN, rARC, and cARC, four images were collected, and five images were collected in the mARC. The abundance of ESR1 mRNA was determined by establishing the number of objects (silver grains) present in a region of interest (ROI) within each image collected. The NIS-Elements software (Nikon) was used for image analysis using procedures similar to those used previously in our laboratory (15). A threshold signal was established and applied to all images before the number of objects was determined in the standardized ROI. The number of objects, which directly corresponded to the number of silver grains present, was recorded within each ROI. Additional images encompassing a neuronal fiber bundle present in the section (i.e., anterior commissure for POA images and fornix for hypothalamic images) were captured and used to determine the background number of silver grains present in each section. Background counts were used to adjust data obtained for each area investigated. To ensure that using a fiber bundle for background correction did not artificially lower the background, we compared the number of objects (silver grains) in a fiber bundle or cell-rich area in one section containing the mARC from three OVX and three OVX+E ewes. The fornix was used as the fiber bundle to determine background, and a cell-rich area in the lateral hypothalamic area, an area where there are little to no cells containing ESR1 (16, 18, 19), was used. Additionally, we also captured images from an mARC section from our sense control slide, because these sections should contain only background silver grains and not ESR1. There was no difference in the number of silver grains detected in a fiber bundle or cell-rich area in either OVX (fiber bundle, 195 ± 45 silver grains vs cell-rich area, 224 ± 5 silver grains) or OVX+E ewes (fiber bundle, 233 ± 90 silver grains vs cell-rich area, 225 ± 55 silver grains), and background in these animals was comparable to background measured in the sense control section (fiber bundle, 175 silver grains; cell-rich area, 202 silver grains). Therefore, fiber bundles were used for background correction in all analyses. ESR1 mRNA abundance per kisspeptin neuron In the POA/PeV and ARC, the number of kisspeptin neurons was determined. Four sections within the POA (including two sections at the level of the OVLT), three through the rARC, four through the mARC, and two through the cARC were selected. Bright-field images were captured at ×40 magnification from 20 kisspeptin neurons in at least three representative sections of the POA, 15 kisspeptin neurons in at least three representative sections of the rARC, 30 kisspeptin neurons in at least four representative sections of the mARC, and 15 kisspeptin neurons in at least two representative sections of the cARC of each ewe. A threshold signal was established and applied to all images and an ROI of 20 µm in diameter was placed over a kisspeptin neuron identified in the image. The number of objects representing the area covered by silver grains was determined in the standardized ROI and recorded. Fifteen ROIs of 20 µm in diameter were placed randomly in an image captured from a neuronal fiber bundle present in the section (i.e., anterior commissure and fornix) to determine background number of silver grains in each section. The average number of silver grains determined in these 15 ROIs was used to adjust the data obtained from images of each neuron. Kisspeptin neurons containing fivefold or more the number of silver grains determined in the background image were identified as ESR1-expressing kisspeptin neurons. ESR1 content per kisspeptin neuron To determine the percentage of kisspeptin neurons in the POA and ARC that contained ESR1, a virtual slide microscope (VS120; Olympus, Tokyo, Japan) was used. After all sections were scanned and digitized using the ×20 objective, four sections within the POA (including two sections at the level of the OVLT), three through the rARC, four through the mARC, and two through the cARC were analyzed using OlyVIA software from Olympus to determine the percentage of kisspeptin neurons that contained ESR1 protein. Hormone assays Concentrations of LH in plasma samples were determined by a double antibody radioimmunoassay previously validated and reported from this laboratory (20). Intra-assay and interassay coefficients of variation were 11.60% and 19.96%, respectively. Statistical analysis Frequency and amplitude of LH pulses were determined using the Pulse4 pulse-detection algorithm available within the Pulse XP program (21). Adjusted ESR1 expression data were transformed to the log10, and the percentage of kisspeptin neurons expressing ESR1 was transformed using the arcsine square root method. Normalized ESR1 expression, the number of kisspeptin neurons, the transformed percentage of kisspeptin neurons expressing ESR1, the mean adjusted number of silver grains per kisspeptin neuron, and the percentage of kisspeptin neurons that contained ESR1 were analyzed by a t test (JM Pro 12; SAS Institute, Cary, NC). Mean concentrations of LH and the amplitude of LH pulses were analyzed using a mixed model analysis for repeated measures (Proc Mixed; SAS Institute) to assess main effects (treatment) and the treatment by time interaction. Ewe ID was the random effect, time was the repeated variable, and ewe ID was the subject. The frequency of LH pulses between OVX and OVX+E lambs was compared using a Wilcoxon–Mann–Whitney test. Means for LH variables are reported as least squares means [± standard error of the mean (SEM)]. Because the frequency of LH pulses was highly variable among ewe lambs in the OVX+E group, lambs were reassigned to one of three separate groups based on the number of LH pulses detected in 12 hours as follows: (1) low frequency (LF; 1 or 2 pulses per 12 hours; n = 3), (2) moderate frequency (MF; 6 or 7 pulses per 12 hours; n = 6), and (3) high frequency (HF; 10 or more pulses per 12 hours; n = 5) and post hoc analyses were performed. Mean concentrations of LH and pulse amplitude data were reanalyzed using Proc Mixed for repeated measures, with main effects of group (OVX, LF, MF, and HF) and the group by time interaction. When significant differences were observed, a Tukey post hoc test was used to compare means. The frequency of LH pulses among the groups was compared as described above using a Wilcoxon–Mann–Whitney test. All other tissue-related data were also reanalyzed based on pulse frequency category using a one-way analysis of variance (ANOVA). The main source of variation was group (OVX, LF, MF, and HF). When significant differences were observed in the ANOVA, a Tukey post hoc test was used to compare means between groups. Results Mean (±SEM) body weight of ewe lambs at 30 weeks of age was 50 ± 1.5 kg and did not differ between OVX and OVX+E ewe lambs. Mean circulating concentrations of LH were greater (P < 0.0001) in OVX (5.5 ± 0.5 ng/mL) than in OVX+E lambs (2.2 ± 0.4 ng/mL). This was accompanied by a greater overall frequency (14.6 ± 1.6 vs 7.8 ± 1.2 pulses per12 hours (P < 0.001) of LH pulses in OVX than in OVX+E lambs with no differences in LH pulse amplitude between the groups (4.0 ± 0.5 ng/mL vs 3.0 ± 0.5 ng/mL). However, lambs in the OVX+E group exhibited significant variability in the frequency of LH pulses, ranging from 1 to 20 pulses per 12 hours. Therefore, in a post hoc analysis, the OVX+E group was repartitioned into three groups based on the number of LH pulses as follows: (1) LF (1 to 2 pulses per 12 hours; n = 3), (2) MF (6 to 7 pulses per 12 hours; n = 6), and (3) HF (10 to 20 pulses per 12 hours; n = 5). Patterns of LH release during the 12-hour sampling period from one representative lamb in each group are presented in Fig. 1A. Analysis indicated that the frequency of LH pulses in OVX and OVX+E HF lambs was similar, both of which were greater (P < 0.0001) than in MF and LF groups, which also differed (Fig. 1B). Differences in concentrations of LH favored OVX over all other groups, and concentrations of LH in HF were greater than LF (P < 0.05; Fig. 1B). There were no differences in LH pulse amplitude between any of the groups. Figure 1. View largeDownload slide (A) Patterns of LH release in representative OVX and OVX+E lambs receiving an estradiol implant exhibiting HF, MF, or LF release. Detected pulses are indicated with asterisks. (B) Least squares mean (±SEM) concentrations of plasma LH and frequency and amplitude of LH pulses in OVX lambs and in OVX+E lambs exhibiting HF, MF, or LF of LH pulses. Mean LH concentrations and amplitude of LH pulses were analyzed using a mixed model analysis for repeated measures; frequency of LH pulses was compared using a Wilcoxon–Mann–Whitney test. Means with different superscripts differ (P < 0.05). Figure 1. View largeDownload slide (A) Patterns of LH release in representative OVX and OVX+E lambs receiving an estradiol implant exhibiting HF, MF, or LF release. Detected pulses are indicated with asterisks. (B) Least squares mean (±SEM) concentrations of plasma LH and frequency and amplitude of LH pulses in OVX lambs and in OVX+E lambs exhibiting HF, MF, or LF of LH pulses. Mean LH concentrations and amplitude of LH pulses were analyzed using a mixed model analysis for repeated measures; frequency of LH pulses was compared using a Wilcoxon–Mann–Whitney test. Means with different superscripts differ (P < 0.05). Overall ESR1 mRNA abundance in the POA and hypothalamus Accumulation of silver grains following in situ hybridization for detection of ESR1 mRNA was observed throughout the POA and hypothalamus (Fig. 2). There were no differences between OVX and OVX+E lambs in the number of silver grains per ROI in the POA (937 ± 97), PeV (724 ± 53), SON (1253 ± 112), PVN (689 ± 72), VMH (559 ± 61), VL-VMH (829 ± 83), and cARC (697 ± 114). In the mARC, OVX lambs exhibited greater ESR1 mRNA abundance (P < 0.05) than did OVX+E lambs, albeit this difference was small (Fig. 2C). There was also a trend (P < 0.08) for OVX lambs to exhibit greater ESR1 mRNA abundance in the rARC than OVX+E lambs (Fig. 2C). No differences were observed among HF, MF, and LF OVX+E lambs in any of the areas studied. Figure 2. View largeDownload slide (A) Detection of ESR1 mRNA in the hypothalamus. Drawing of a hypothalamic section containing the VL-VMH and mARC. (B) Low-magnification image of the boxed area in (A) depicting signal at the level of the mARC and a portion of the VL-VMH. (C) Normalized mean (±SEM) number of silver grains in the rostral (rARC, top panel), middle (mARC, middle panel) and caudal (cARC, bottom panel) portions of the ARC in OVX and OVX+E lambs. The number of silver grains in the rARC (top panel) of OVX lambs tended to be greater (t test; #P < 0.08) than in the OVX+E lambs. The number of silver grains in the mARC (middle panel) of OVX lambs was greater (t test; *P < 0.05) than in OVX+E lambs. No differences were observed in the cARC (bottom panel). The asterisk in (B) indicates an artifact associated with meninges. 3V, third ventricle; fx, fornix; pt, pars tuberalis. Figure 2. View largeDownload slide (A) Detection of ESR1 mRNA in the hypothalamus. Drawing of a hypothalamic section containing the VL-VMH and mARC. (B) Low-magnification image of the boxed area in (A) depicting signal at the level of the mARC and a portion of the VL-VMH. (C) Normalized mean (±SEM) number of silver grains in the rostral (rARC, top panel), middle (mARC, middle panel) and caudal (cARC, bottom panel) portions of the ARC in OVX and OVX+E lambs. The number of silver grains in the rARC (top panel) of OVX lambs tended to be greater (t test; #P < 0.08) than in the OVX+E lambs. The number of silver grains in the mARC (middle panel) of OVX lambs was greater (t test; *P < 0.05) than in OVX+E lambs. No differences were observed in the cARC (bottom panel). The asterisk in (B) indicates an artifact associated with meninges. 3V, third ventricle; fx, fornix; pt, pars tuberalis. Number and distribution of kisspeptin neurons in the POA and ARC The number of kisspeptin-immunoreactive neurons detected in the POA of OVX lambs was limited, with no or only a few (up to four) neurons observed in each lamb. Therefore, no comparisons between OVX and OVX+E lambs were performed for the POA. In contrast to OVX lambs, numerous kisspeptin neurons were observed in the POA of OVX+E lambs (257 ± 37). However, no differences in the number of kisspeptin-immunoreactive neurons in the POA were observed among OVX+E lambs in HF, MF, and LF groups. Kisspeptin-immunoreactive neurons were readily detected in the ARC (Fig. 3A) in substantial numbers in both OVX and OVX+E lambs. The number of kisspeptin neurons was greater (P < 0.001) in OVX than in OVX+E lambs in the rARC and mARC (Fig. 4). In the cARC, the number of kisspeptin neurons was also greater in OVX than OVX+E lambs, but the difference was not statistically significant (P < 0.11; Fig. 4C). There were no differences among OVX+E lambs in HF, MF, and LF groups for the number of kisspeptin-immunoreactive neurons in any of the three subdivisions of the ARC. Figure 3. View largeDownload slide Images of a section at the level of the ARC processed for dual-label detection of ESR1-containing and kisspeptin-immunoreactive neurons. (A) Low-magnification image depicting kisspeptin-immunoreactive neurons (brown) and cells stained nonspecifically with cresyl violet (purple) in the mARC. (B) High-magnification image of two kisspeptin neurons shown in (A) (arrows) exhibiting silver grain accumulation over the brown-stained cell body and proximal dendrite. (C) High-magnification image of a kisspeptin neuron shown in (A) (arrowhead) exhibiting only few silver grains accumulated over the brown-stained cell body and proximal dendrites. 3V, third ventricle. Figure 3. View largeDownload slide Images of a section at the level of the ARC processed for dual-label detection of ESR1-containing and kisspeptin-immunoreactive neurons. (A) Low-magnification image depicting kisspeptin-immunoreactive neurons (brown) and cells stained nonspecifically with cresyl violet (purple) in the mARC. (B) High-magnification image of two kisspeptin neurons shown in (A) (arrows) exhibiting silver grain accumulation over the brown-stained cell body and proximal dendrite. (C) High-magnification image of a kisspeptin neuron shown in (A) (arrowhead) exhibiting only few silver grains accumulated over the brown-stained cell body and proximal dendrites. 3V, third ventricle. Figure 4. View largeDownload slide Mean (±SEM) number of kisspeptin-immunoreactive neurons in the (A) rARC, (B) mARC, and (C) cARC. The mean number of kisspeptin neurons was greater (t test; **P < 0.001) in the rARC and mARC of OVX lambs than in OVX+E lambs. Figure 4. View largeDownload slide Mean (±SEM) number of kisspeptin-immunoreactive neurons in the (A) rARC, (B) mARC, and (C) cARC. The mean number of kisspeptin neurons was greater (t test; **P < 0.001) in the rARC and mARC of OVX lambs than in OVX+E lambs. ESR1 mRNA abundance in kisspeptin neurons A considerable number of kisspeptin neurons of both POA and ARC populations were observed to contain ESR1 (Fig. 3B), although many were also observed to exhibit no meaningful accumulation of silver grains (Fig. 3C). In the POA of OVX+E lambs, the percentage of ESR1-positive kisspeptin neurons was 58% ± 7%, and the percentage of ESR1-positive kisspeptin neurons did not differ among OVX+E lambs in the HF, MF, and LF groups. The mean number of silver grains per ESR1-positive kisspeptin neuron in the POA of OVX+E lambs also did not differ among the HF, MF, and LF groups (9.4 ± 0.5). In the ARC, the percentage of ESR1-positive kisspeptin neurons was 50%, 44%, and 37% for the rARC, mARC, and cARC, respectively, and did not differ between OVX and OVX+E lambs. However, the percentage of ESR1-positive neurons in the mARC was greater (P < 0.05) in HF than in LF lambs (Fig. 5) and tended to be greater (P < 0.06) than the MF group (Fig. 5). The mean percentage of ESR1-positive kisspeptin neurons located in the mARC in OVX lambs did not differ from HF and MF groups but tended to be greater (P < 0.06) than in the LF group (Fig. 5). The mean number of silver grains per kisspeptin neuron also tended to be greater (P < 0.07) in the rARC and mARC of OVX than in OVX+E lambs, but did not differ in the cARC (Fig. 6). When comparing HF, MF, and LF groups, there were no differences in the mean number of silver grains per kisspeptin neuron. Figure 5. View largeDownload slide Mean (±SEM) percentage of ESR1-positive kisspeptin neurons in the mARC of OVX lambs and OVX+E lambs exhibiting HF, MF, and LF of LH pulses (one-way ANOVA; #P < 0.06, *P < 0.05). Figure 5. View largeDownload slide Mean (±SEM) percentage of ESR1-positive kisspeptin neurons in the mARC of OVX lambs and OVX+E lambs exhibiting HF, MF, and LF of LH pulses (one-way ANOVA; #P < 0.06, *P < 0.05). Figure 6. View largeDownload slide Mean (±SEM) number of grains per kisspeptin neuron in OVX and OVX+E lambs in the (A) rARC, (B) mARC, and (C) cARC. The average number of grains per kisspeptin neuron tended (t test; #P < 0.07) to be greater in OVX lambs than in OVX+E lambs in the rARC and mARC. Figure 6. View largeDownload slide Mean (±SEM) number of grains per kisspeptin neuron in OVX and OVX+E lambs in the (A) rARC, (B) mARC, and (C) cARC. The average number of grains per kisspeptin neuron tended (t test; #P < 0.07) to be greater in OVX lambs than in OVX+E lambs in the rARC and mARC. Colocalization of kisspeptin neurons with ESR1 protein A high percentage of kisspeptin neurons in both the POA and ARC were found to contain ESR1 (Fig. 7). In the POA of OVX+E lambs, 90% ± 2% of kisspeptin neurons were found to contain ESR1 but did not differ among OVX+E HF, MF, and LF groups. Whereas a high percentage of kisspeptin neurons in the ARC of both OVX and OVX+E lambs contained ESR1 (82.6% ± 1.3%), a greater percentage (P < 0.05) of kisspeptin neurons in OVX lambs contained ESR1 compared with OVX+E lambs in the rARC, mARC, and cARC (Fig. 8). The mean percentage of kisspeptin neurons that contained ESR1 did not differ among OVX+E HF, MF, and LF groups. Figure 7. View largeDownload slide High-magnification image depicting kisspeptin-immunoreactive neurons (brown) and ESR1 (black) in the mARC. Representative kisspeptin neurons that contain ESR1 are indicated by white arrows, and representative kisspeptin neurons that do not contain ESR1 are indicated by black arrows. Figure 7. View largeDownload slide High-magnification image depicting kisspeptin-immunoreactive neurons (brown) and ESR1 (black) in the mARC. Representative kisspeptin neurons that contain ESR1 are indicated by white arrows, and representative kisspeptin neurons that do not contain ESR1 are indicated by black arrows. Figure 8. View largeDownload slide Mean (±SEM) percentage of kisspeptin neurons containing ESR1 in OVX and OVX+E lambs in the (A) rARC, (B) mARC, and (C) cARC. The percentage of kisspeptin neurons that contained ESR1 was greater in OVX than in OVX+E lambs in the rARC (t test; *P < 0.05), mARC (t test; **P < 0.001), and cARC (t test; *P < 0.05). Figure 8. View largeDownload slide Mean (±SEM) percentage of kisspeptin neurons containing ESR1 in OVX and OVX+E lambs in the (A) rARC, (B) mARC, and (C) cARC. The percentage of kisspeptin neurons that contained ESR1 was greater in OVX than in OVX+E lambs in the rARC (t test; *P < 0.05), mARC (t test; **P < 0.001), and cARC (t test; *P < 0.05). Discussion The results of experiments reported in this study indicate that, although estradiol reduces the number of kisspeptin neurons in prepubertal/peripubertal ewe lambs, the escape from estradiol negative feedback, although clearly a fundamental element of pubertal maturation in sheep (2, 8, 22, 23), is not associated with declines in ESR1 expression or ESR1 protein in kisspeptin neurons. Indeed, as the escape from estradiol negative feedback became evident in HF and MF females in the present experiment, ESR1 expression in kisspeptin neurons increased relative to LF lambs and did not differ from OVX only females. Similarly, abundant ESR1 protein in kisspeptin neurons was observed in all groups, with the greatest amount observed in OVX lambs. Studies in mice have indicated that ESR1 is the major type of estrogen receptor mediating estradiol negative feedback effects on GnRH secretion (9), and neurons located in the ARC that contain ESR1 are essential for communicating estradiol negative feedback (24). In a study using intact heifers, a reduction in the overall number of estradiol receptors in the anterior and mediobasal hypothalamus was observed to occur as heifers approached puberty (25). A similar decline in the number of estrogen receptors in the hypothalamus was also detected during juvenile development in female rats (26). Because both studies used estradiol-binding assays of hypothalamic extracts, the specific type of estrogen receptor investigated, as well as the regional location within the hypothalamus in which receptors were present, was not determined. Therefore, a direct comparison of those data with changes in overall ESR1 mRNA abundance observed in the present study should be done with caution. Nevertheless, the results of the present study in ewe lambs suggest that the decrease in estradiol binding in the hypothalamus reported during reproductive maturation in rats (26) and heifers (25) are not negatively associated with changes in overall ESR1 mRNA abundance or ESR1 protein. The effects of estradiol on the regulation of ESR1 mRNA abundance in specific hypothalamic areas appear more consistent among the various studies reported. Estrogen receptor mRNA in the VMH and ARC decreases with estradiol treatment in mature, ovariectomized rats (27). Although we did not observe changes in ESR1 mRNA in the VMH between OVX and OVX+E lambs, estradiol treatment decreased the overall abundance of ESR1 mRNA in the ARC. Whether the difference in ESR1 expression in the VMH reflects distinction among species, or represents differences in the animal model as related to the maturation of neuroendocrine function, is unclear. Nevertheless, the consistency between studies on the effects of estradiol in the regulation of ESR1 mRNA abundance in the ARC indicates the relevance of this hypothalamic area for estradiol’s control of reproductive function in females. The presence or absence of estradiol has also been shown to affect ESR1 immunoreactivity. In rats, estradiol downregulates estrogen receptor immunoreactivity in the medial preoptic nucleus (28). The density of ESR1-immunoreactive neurons was lower in the AVPV, VL-VMH, and ARC of ovariectomized rats treated with estradiol benzoate than in untreated rats (29). Abundance of Kiss1 mRNA has also been demonstrated to be influenced by estradiol. In mature ewes, Kiss1 expression in the ARC is increased owing to ovariectomy, and estradiol replacement decreases Kiss1 mRNA in the ARC to levels similar to those of intact ewes (30). Likewise, ovariectomy increases the number of immunoreactive kisspeptin neurons in the ARC (31). In the present study, we observed that the number of kisspeptin neurons in the rARC and mARC was greater in ovariectomized ewe lambs that did not receive an estradiol implant. This observation is consistent with the findings of the study reported by Nestor et al. (32) in which the number of kisspeptin neurons increased in the ARC of prepubertal ewes that were ovariectomized compared with intact ewes. The study by Nestor et al. (32) also reported that the number of kisspeptin neurons was greater in postpubertal compared with prepubertal intact ewes. Because the frequency of LH pulses increases as ewes approach maturity (33), it was plausible to expect that the number of kisspeptin neurons would increase in the ARC as the frequency of LH pulses increased in peripubertal lambs of the present study. An earlier study in our laboratory indicating that Kiss1 mRNA abundance increases in the ARC in association with increased LH pulsatility in ovariectomized, estradiol-treated lambs (15) supports this hypothesis. However, in the present study, no differences in the number of kisspeptin neurons were detected in the ARC among groups of ovariectomized, estradiol-implanted lambs exhibiting high, moderate, or low LH pulsatility. Thus, immediate changes in kisspeptin synthesis and accumulation in ARC neurons do not appear to clearly precede the decrease in the sensitivity to estradiol negative feedback during reproductive maturation in sheep. Estradiol likely regulates Kiss1 mRNA abundance directly because ESR1 is detected in kisspeptin neurons in sheep (11). Therefore, it has been proposed that kisspeptin neurons mediate the estradiol feedback regulation of GnRH and LH secretion. Research in mice has demonstrated that Esr1 in kisspeptin neurons is critical for the control of LH pulsatility and reproductive function. Selective ablation of Esr1 in kisspeptin neurons in female mice increases Kiss1 mRNA in the ARC and circulating concentrations of LH (13). Additionally, whereas these mice exhibit increased concentrations of LH and advanced vaginal opening, normal estrous cyclicity is absent. The ability of estradiol to suppress LH secretion and Kiss1 mRNA abundance in prepubertal but not adult female mice is also dependent on the presence of Esr1 in kisspeptin neurons (14, 34). These observations support the role for Esr1 in kisspeptin neurons as important for signaling estradiol feedback control of gonadotropin release in mice (13, 14). Therefore, even though only small differences in overall ESR1 expression in the ARC between OVX and OVX+E prepubertal ewes were observed in this study, it is seemingly plausible to expect such changes in a neuronal population as critically important to reproduction (e.g., kisspeptin neurons). We hypothesized that downregulation of ESR1 in kisspeptin neurons could facilitate the increased kisspeptin synthesis and release during the pubertal transition and downstream stimulation of GnRH secretory activity. However, contrary to our hypothesis, we observed that the percentage of kisspeptin neurons in the ARC expressing ESR1 mRNA was greater in OVX+E lambs exhibiting a high frequency of LH pulses than in less reproductively mature lambs. Thus, the ability of estradiol to inhibit ESR1 transcription in kisspeptin neurons in the ARC appears less in lambs exhibiting a more advanced stage of reproductive maturation and decreased sensitivity to estradiol negative feedback. Because the percentage of kisspeptin neurons in the ARC that expressed ESR1 mRNA increased instead of decreased in OVX+E lambs that exhibited increased LH pulsatility, we also examined the percentage of kisspeptin neurons that contained ESR1 protein in the ARC of OVX and OVX+E lambs. Regardless of treatment group, we found that a greater percentage of kisspeptin neurons (>90%) contained ESR1 protein compared with ESR1 mRNA (50%). These differences are likely due to the different thresholds of detection between the techniques of in situ hybridization and immunocytochemistry. In keeping with previous reports from this laboratory (15, 35), kisspeptin neurons in the present experiment were required to contain fivefold more silver grains than the background signal to be considered as expressing ESR1 mRNA. This is a relatively stringent threshold. If this threshold is decreased to threefold the background, the percentage of kisspeptin cells that are also considered positive for ESR1 mRNA increases to ∼85%, comparable to that estimated for containing ESR1 protein. The percentage of kisspeptin neurons that contained ESR1 protein remained slightly but significantly greater in OVX lambs compared with OVX+E lambs, and there were no differences among HF, MF, and LF groups. Because the presence of estradiol is known to decrease the number of kisspeptin neurons in the ARC of prepubertal ewe lambs (32), the difference observed in the percentage of kisspeptin neurons that contain ESR1 for OVX and OVX+E ewes may be due to a decrease in the number of kisspeptin neurons. Therefore, although estradiol decreases the percentage of kisspeptin neurons that contain ESR1 protein in the ARC of lambs, other mechanisms appear to be more directly involved in controlling the escape from estradiol negative feedback. It is also possible that the number of ESR1 receptors present within an individual kisspeptin neuron is changing as LH pulse frequency increases. However, the use of immunocytochemistry to detect kisspeptin and ESR1 protein does not provide a strategy for answering this question. Immunocytochemistry is a semiquantitative technique whereby cells are classified as either expressing or not expressing a protein. A more quantitative technique would be needed to answer this question. In addition to participating in the communication of estradiol negative feedback to GnRH neurons, kisspeptin neurons are likely to also be involved in the transmission of estradiol positive feedback. In rodents, kisspeptin neurons located in the POA appear to play such a role (36–40). However, the location of kisspeptin neurons contributing to estradiol positive feedback in sheep is more ambiguous than in rodents. Whereas the expression of KISS1 in the POA increases during the late follicular phase in ewes (41), as well as during juvenile development (11, 15, 41), the percentage of kisspeptin neurons in both the POA and ARC exhibiting Fos activity (a marker of neuronal activation) is observed to increase during the preovulatory surge of LH in mature ewes (42). In contrast, Hoffman et al. (43) observed Fos labeling only in kisspeptin neurons in the POA during the preovulatory surge in ewes. Therefore, the role of kisspeptin neurons in the ARC in mediating estradiol stimulation of the preovulatory LH surge in the ewe requires further elucidation. In the absence of estradiol implants, few kisspeptin neurons were observed in the POA of OVX lambs. This is in agreement with previous reports in sheep (32) and mice (13, 44). The previous observation that the numbers of kisspeptin neurons do not change as the frequency of LH pulses increases in 30-week-old ewe lambs is also in agreement with our previous study (15). Although estradiol is required for activation of KISS1 mRNA in the POA, estradiol does not appear to regulate ESR1 mRNA abundance in kisspeptin neurons in the POA in a manner similar to that in the ARC. However, the percentage of kisspeptin neurons that contained ESR1 protein was high in both the POA and ARC. A much greater percentage of kisspeptin neurons in the POA was found to contain ESR1 in this study when compared with a previous study conducted in adult ewes in the luteal phase (11). It is possible that these discrepancies were caused by differences in endocrine status or age of the animals used in the two studies. To our knowledge, no studies have been conducted to determine whether the percentage of kisspeptin neurons that contain ESR1 changes throughout the estrous cycle in female sheep. In summary, contrary to our hypothesis, the increase in LH pulsatility in estradiol-treated, maturing ewe lambs was associated with enhanced ESR1 mRNA abundance in kisspeptin neurons in the ARC, and the absence of estradiol in OVX ewe lambs was associated with the greatest ESR1 abundance and percentage of kisspeptin neurons containing ESR1 protein in the ARC. Therefore, changes in the expression of ESR1, particularly in kisspeptin neurons in the ARC, fail to explain the pubertal escape from estradiol negative feedback in ewe lambs. Appendix. Antibody Table Peptide/Protein Target  Antigen Sequence  Name of Antibody  Manufacturer, Catalog No, and/or Name of Individual Providing the Antibody  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  Kisspeptin  Peptide from mouse kisspeptin 10  Anti-kisspeptin antibody  Gifted by A. Caraty  Rabbit; polyclonal  1:75,000  AB_2622231  Kisspeptin  Peptide from mouse kisspeptin 10  Anti-kisspeptin antibody  Millipore, AB9754  Rabbit; polyclonal  1:2000  AB_229652  ERα  Recombinant human estrogen receptor protein  ERα (clone 1D5)  Dako, M7047  Mouse; monoclonal  0.388888889  AB_2101946  LH  Ovine LH  Anti-oLH ab  A.F. Parlow, National Pituitary Hormone Program  Rabbit  1:125,000  AB_2716713  Peptide/Protein Target  Antigen Sequence  Name of Antibody  Manufacturer, Catalog No, and/or Name of Individual Providing the Antibody  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  Kisspeptin  Peptide from mouse kisspeptin 10  Anti-kisspeptin antibody  Gifted by A. Caraty  Rabbit; polyclonal  1:75,000  AB_2622231  Kisspeptin  Peptide from mouse kisspeptin 10  Anti-kisspeptin antibody  Millipore, AB9754  Rabbit; polyclonal  1:2000  AB_229652  ERα  Recombinant human estrogen receptor protein  ERα (clone 1D5)  Dako, M7047  Mouse; monoclonal  0.388888889  AB_2101946  LH  Ovine LH  Anti-oLH ab  A.F. Parlow, National Pituitary Hormone Program  Rabbit  1:125,000  AB_2716713  View Large Abbreviations: ANOVA analysis of variance ARC arcuate nucleus cARC caudal ARC cDNA complementary DNA DAB 3,3′-diaminobenzidine ESR1 estrogen receptor α HF high frequency LF low frequency LH luteinizing hormone mARC middle ARC MF moderate frequency mRNA messenger RNA NGS normal goat serum OVLT organum vasculosum of the lamina terminalis OVX ovariectomy without estradiol implant OVX+E ovariectomy with estradiol implant PBS phosphate-buffered saline PBSTX phosphate-buffered saline containing 0.4% Triton X-100 PCR polymerase chain reaction PeV periventricular nucleus POA preoptic area PVN paraventricular nucleus rARC rostral ARC ROI region of interest RRID Research Resource Identifier SEM standard error of the mean SON supraoptic nucleus SSC saline sodium citrate VL-VMH ventrolateral–ventromedial hypothalamus VMH ventromedial hypothalamus. 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Pubertal Escape From Estradiol Negative Feedback in Ewe Lambs Is Not Accounted for by Decreased ESR1 mRNA or Protein in Kisspeptin Neurons

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0013-7227
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1945-7170
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10.1210/en.2017-00593
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Abstract

Abstract In this study, we investigated whether decreased sensitivity to estradiol negative feedback is associated with reduced estrogen receptor α (ESR1) expression in kisspeptin neurons as ewe lambs approach puberty. Lambs were ovariectomized and received no implant (OVX) or an implant containing estradiol (OVX+E). In the middle arcuate nucleus (mARC), ESR1 messenger RNA (mRNA) was greater in OVX than OVX+E lambs but did not differ elsewhere. Post hoc analysis of luteinizing hormone (LH) secretion from OVX+E lambs revealed three patterns of LH pulsatility: low [1 to 2 pulses per 12 hours; low frequency (LF), n = 3], moderate [6 to 7 pulses per 12 hours; moderate frequency (MF), n = 6], and high [>10 pulses per 12 hours; high frequency (HF), n = 5]. The percentage of kisspeptin neurons containing ESR1 mRNA in the preoptic area did not differ among HF, MF, or LF groups. However, the percentage of kisspeptin neurons containing ESR1 mRNA in the mARC was greater in HF (57%) than in MF (36%) or LF (27%) lambs and did not differ from OVX (50%) lambs. A higher percentage of kisspeptin neurons contained ESR1 protein in all regions of the arcuate nucleus (ARC) in OVX compared with OVX+E lambs. There were no differences in ESR1 protein among the HF, MF, or LF groups in the preoptic area or ARC. Contrary to our hypothesis, increases in LH pulsatility were associated with enhanced ESR1 mRNA abundance in kisspeptin neurons in the ARC, and absence of estradiol increased the percentage of kisspeptin neurons containing ESR1 protein in the ARC. Therefore, changes in the expression of ESR1, particularly in kisspeptin neurons in the ARC, do not explain the pubertal escape from estradiol negative feedback in ewe lambs. Puberty is a well-characterized reproductive event that has been investigated extensively in female mammals. As female sheep approach reproductive maturation, the frequency of luteinizing hormone (LH) pulses increases (1–3). It is known that activation of GnRH neurons in the preoptic area and hypothalamus, as well as secretion of GnRH into the hypothalamic–hypophyseal portal vasculature, is critical for the initiation of the pubertal pattern of LH release (4, 5). However, the neuroendocrine mechanisms involved in controlling the onset of high-frequency GnRH/LH release are still unclear. Whereas a decline in estradiol negative feedback has not been definitively shown to play a major role in pubertal onset across all species, including nonhuman primates (6) and rats (7), a decline in estradiol negative feedback is absolutely essential for the increase in GnRH and LH pulse frequency associated with the initiation of puberty in sheep (2, 8). However, GnRH neurons do not contain estrogen receptor α (ESR1), which is thought to be the major estrogen receptor mediating estradiol’s regulation of GnRH secretion (9). Another estrogen receptor, estrogen receptor β, is expressed in a subpopulation of GnRH neurons in sheep (10), but it does not appear to be important for the control of reproductive neuroendocrine processes (9). Therefore, it is hypothesized that afferent neurons containing ESR1 in the hypothalamus mediate the feedback effects of estradiol on GnRH neurons. Kisspeptin neurons contain ESR1 (11), and kisspeptin stimulates GnRH secretion (12). The absence of Esr1 in kisspeptin neurons has also been observed to prematurely activate pulsatile secretion of LH, increase Kiss1 messenger RNA (mRNA) abundance, and advance the day of vaginal opening in female mice (13, 14). Therefore, elucidating the control of ESR1 expression in kisspeptin neurons could reveal important information regarding estradiol’s role in controlling GnRH secretion and reproductive cyclicity. The present study investigated the hypothesis that decreased sensitivity to estradiol negative feedback and subsequent increases in LH pulsatility in peripubertal female sheep are associated with reduced ESR1 mRNA abundance and protein content in kisspeptin neurons. Materials and Methods Animal experiments were conducted at the Texas A&M University Nutrition and Physiology Center located in the O.D. Butler Animal Science Teaching and Research Complex in College Station, Texas. All procedures used in these studies were approved by the Institutional Agricultural Animal Care and Use Committee of the Texas A&M University and followed National Institutes of Health guidelines for use of animals in research. Animals and experimental procedures Spring-born ewe lambs (n = 21) were ovariectomized at ∼24 weeks of age. Lambs were housed in individual pens and fed ad libitum a commercial diet formulated to meet National Research Council recommendations for growing lambs. At the time of ovariectomy, ewe lambs were selected randomly to receive either no implant (OVX; n = 7) or a subcutaneous implant containing crystalline estradiol (Sigma-Aldrich, St. Louis, MO) (OVX+E; n = 14). The estradiol implants were designed to produce circulating concentrations of estradiol of ∼1 to 2 pg/mL and successfully maintain estradiol negative feedback on gonadotropin release in ovariectomized ewe lambs (8), as reported previously from this laboratory (15). At 30 weeks of age, a time when ewes are approaching pubertal transition, a catheter (16-gauge by 3 inches in polyurethane; Jorgensen Laboratories, Loveland, CO) was inserted into a jugular vein 24 hours before initial blood sampling. The next day, lambs were restrained loosely in their home pen with a halter and blood samples (5 mL) were collected remotely every 10 minutes for 12 hours using an extension connected to the jugular catheter. Lambs were acclimated to experimental conditions of blood collection for 5 days before blood sampling and were housed adjacent to other sheep at all times. Immediately after collection, blood samples were placed in tubes containing 50 µL of a solution of heparin (3000 U/mL) and 5% EDTA and mixed gently. Samples were placed immediately on ice and centrifuged at 2200 × g for 20 minutes at 4°C within 2 hours of collection. Plasma was collected and stored at −20°C until processing to determine concentrations of LH. On the day after intensive collection of blood samples, ewe lambs were euthanized with an overdose of pentobarbital (Beuthanasia-D Special; Schering-Plough, Union, NJ), and heads were perfused with a solution containing 4% paraformaldehyde. Brains were dissected from the cranium and a block of tissue containing the septum, preoptic area (POA), and hypothalamus was collected and placed in 4% paraformaldehyde at 4°C for 48 hours, with paraformaldehyde solution replaced after 24 hours. After paraformaldehyde incubation, tissue blocks were placed in a 0.1 M phosphate-buffered solution containing 30% sucrose at 4°C for at least 7 days. Tissue processing Tissue blocks were sectioned in coronal sections of 50 µm using a freezing microtome (Microm HM430; Microm International, Walldorf, Germany). Sections were then stored at −20°C in a cryopreservative solution until processed for double-label in situ hybridization and immunocytochemistry for ESR1 and kisspeptin or double-label immunocytochemistry for ESR1 and kisspeptin. Double-label in situ hybridization and immunocytochemistry To detect ESR1 mRNA, sense and antisense radiolabeled complementary RNA probes were generated by in vitro transcription of a DNA template containing a 311-bp sequence of ovine ESR1 complementary DNA (cDNA) linked to RNA polymerase promoters. The template was produced by polymerase chain reaction (PCR) from the linearized poER8 plasmid containing a partial ovine ESR1 cDNA provided graciously by Dr. Nancy Ing (Texas A&M University; GenBank accession number U30299.1) (16), following procedures described previously (17). The initial PCR used the primers 5′-CGAGCGGCTATGCGGTG-3′ and 5′-GGCCTGACAGCTCTTCCTTC-3′ to amplify an ESR1-specific sequence. A second reaction was performed using the product of the first reaction as a template and primers tagged with the T3 and T7 RNA polymerase promoter sequences. Primers used in the second reaction were 5′-AATTAACCCTCACTAAAGGGCGAGCGGCTATGCGGTG-3′ (T3 promoter in bold type) and 5′-TAATACGACTCACTATAGGGAGGCCTGACAGCTCTTCCTTC-3′ (T7 promoter in bold type). The PCR product was sequenced and the sequence obtained was aligned to the available Ovis aries genomic database using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The cDNA obtained was 100% identical to the ovine ESR1 cDNA. The in vitro transcription reaction for synthesis of 35S-labeled probes followed procedures described previously (15). Free-floating sections were washed in 0.1 M phosphate buffer 10 times for 6 minutes each. The sections were then placed in a 1% NaBH4 solution for 15 minutes. Sections were washed in 0.1 M phosphate buffer 10 times for 6 minutes each and then washed in 0.1 M triethanolamine twice for 10 minutes. Next, sections were incubated in a 0.25% acetic anhydride solution for 10 minutes before being washed in 2× saline sodium citrate (SSC) three times for 10 minutes. Sections were then hybridized with sense or antisense radiolabeled complementary RNA probes for ESR1 overnight at 55°C. Before hybridization, probes were diluted in hybridization buffer (50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl, 5 mM EDTA, 10 mM NaPO4, Denhardt solution, 10% dextran sulfate, 0.5 mg/ml yeast transfer RNA, and 100 mM dithiothreitol) and denatured at 70°C for 10 minutes. After overnight incubation, the sections were briefly washed four times in 4× SSC and then treated with RNase in a buffer containing Tris-Cl, EDTA, and NaCl for 30 minutes at 37°C. The sections were then incubated in a 1× Tris-Cl, EDTA, and NaCl buffer for 30 minutes at 37°C followed by washes in 2× SSC for 30 minutes at 55°C and 0.2× SSC for 30 minutes at 55°C. After completion of the in situ hybridization procedure, tissue sections were immediately processed for immunodetection of kisspeptin. Sections were washed in phosphate-buffered saline (PBS) four times for 5 minutes and then incubated in PBS containing 1% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity. After further washing, sections were incubated in a solution of PBS containing 0.4% Triton X-100 (PBSTX) and 4% normal goat serum (NGS) for at least 1 hour. Sections were then incubated in a solution containing rabbit anti-kisspeptin antiserum [AC no. 564; 1:75,000; Research Resource Identifier (RRID): AB_2622231; provided by Dr. Alain Caraty, Institut National de la Recherche Agronomique, Nouzilly, France] in PBSTX and 4% NGS for 16 hours. After incubation with the primary antibody, sections were washed and incubated in a solution containing biotinylated goat anti-rabbit IgG (1:400; Vector Laboratories, Burlingame, CA), PBSTX, and 4% NGS for 1 hour. The sections were washed and incubated in a solution containing streptavidin–horseradish peroxidase conjugate (Vectastain Elite ABC; 1:600; Vector Laboratories) for 1 hour. Sections were then washed and incubated in a solution containing 3,3′-diaminobenzidine (DAB; 0.2 mg/mL) with hydrogen peroxide (0.012%; Sigma-Aldrich) in PBS for 10 minutes. The sections were washed again, mounted on slides, and dried at 37°C. Slides were dipped in photographic NBT2 emulsion (Kodak Company, Rochester, NY), dried, and exposed in the dark for 35 days at 4°C. Slides were developed in D-19 developer and sections were counterstained with cresyl violet, dehydrated, and covered with glass slips using DPX (VWR International, Radnor, PA). Double-label immunocytochemistry Similar to the above immunocytochemical protocol, sections were washed in PBS four times for 5 minutes and then incubated in PBS containing 1% hydrogen peroxide for 10 minutes. After further washing, the sections were incubated in a solution of PBS containing 0.4% PBSTX and 4% NGS for at least 1 hour. The sections were then incubated in a solution containing mouse anti-ESR1 antiserum (catalog no. M7047; clone 1D5; RRID: AB_2101946; 1:500; DAKO, Glostrup, Denmark) in PBSTX and 4% NGS for 16 hours. After incubation with the primary antibody, sections were washed and then incubated in a solution containing biotinylated goat anti-mouse IgG (1:400; Vector Laboratories), PBSTX, and 4% NGS for 1 hour. The sections were washed and incubated in a solution containing streptavidin–horseradish peroxidase conjugate (Vectastain Elite ABC; 1:600; Vector Laboratories) for 1 hour. Sections were then washed and incubated in a solution containing DAB (0.2 mg/mL), nickel sulfate (2%; Sigma-Aldrich), and hydrogen peroxide (0.012%; Sigma-Aldrich) in PBS for 10 minutes. The sections were washed and incubated in PBS containing 1% hydrogen peroxide for 10 minutes. After further washing, the sections were incubated in PBSTX and 4% NGS for at least 1 hour. The sections were then incubated in a solution containing rabbit anti-kisspeptin antiserum (catalog no. AB9754; RRID: AB_229652; 1:2000; Millipore, Billerica, MA) in PBSTX and 4% NGS for 16 hours. After incubation with the primary antibody, the sections were washed and then incubated in a solution containing biotinylated goat anti-rabbit IgG (1:400; Vector Laboratories), PBSTX, and 4% NGS for 1 hour. The sections were washed and incubated in a solution containing streptavidin–horseradish peroxidase conjugate (Vectastain Elite ABC; 1:600; Vector Laboratories) for 1 hour. Sections were then washed and incubated in a solution containing DAB (0.2 mg/mL) with hydrogen peroxide (0.012%; Sigma-Aldrich) in PBS for 10 minutes. The sections were washed, mounted on slides, and dried at 37°C. Slides were dehydrated and covered with glass slips using DPX (VWR International). ESR1 mRNA abundance in the POA and hypothalamus Processed slides were coded and tissue sections were analyzed by an observer who was unaware of each animal’s experimental group. Overall ESR1 mRNA abundance in the POA and hypothalamus was determined using a dark- and bright-field microscope (Nikon 80i Eclipse; Nikon, Melville, NY). The anatomical location and distribution of ESR1 mRNA was investigated in the POA, periventricular nucleus (PeV), supraoptic nucleus (SON), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), ventrolateral–ventromedial hypothalamus (VL-VMH), and arcuate nucleus (ARC). For each region examined, four sections within the POA [including two sections at the level of the organum vasculosum of the lamina terminalis (OVLT)], three through the PeV, four through the SON, four through the PVN, two through the rostral ARC (rARC), four through the middle ARC (mARC), two through the caudal ARC (cARC), three through the VMH, and two through the VL-VMH were selected. Sections were selected to represent comparable levels within each area examined. Bright-field images of each region were captured using a ×40 objective and a digital camera (DS-Qi1; Nikon) attached to the microscope. In the SON, VMH, and VL-VMH, three images were collected per section analyzed. In the POA, PeV, PVN, rARC, and cARC, four images were collected, and five images were collected in the mARC. The abundance of ESR1 mRNA was determined by establishing the number of objects (silver grains) present in a region of interest (ROI) within each image collected. The NIS-Elements software (Nikon) was used for image analysis using procedures similar to those used previously in our laboratory (15). A threshold signal was established and applied to all images before the number of objects was determined in the standardized ROI. The number of objects, which directly corresponded to the number of silver grains present, was recorded within each ROI. Additional images encompassing a neuronal fiber bundle present in the section (i.e., anterior commissure for POA images and fornix for hypothalamic images) were captured and used to determine the background number of silver grains present in each section. Background counts were used to adjust data obtained for each area investigated. To ensure that using a fiber bundle for background correction did not artificially lower the background, we compared the number of objects (silver grains) in a fiber bundle or cell-rich area in one section containing the mARC from three OVX and three OVX+E ewes. The fornix was used as the fiber bundle to determine background, and a cell-rich area in the lateral hypothalamic area, an area where there are little to no cells containing ESR1 (16, 18, 19), was used. Additionally, we also captured images from an mARC section from our sense control slide, because these sections should contain only background silver grains and not ESR1. There was no difference in the number of silver grains detected in a fiber bundle or cell-rich area in either OVX (fiber bundle, 195 ± 45 silver grains vs cell-rich area, 224 ± 5 silver grains) or OVX+E ewes (fiber bundle, 233 ± 90 silver grains vs cell-rich area, 225 ± 55 silver grains), and background in these animals was comparable to background measured in the sense control section (fiber bundle, 175 silver grains; cell-rich area, 202 silver grains). Therefore, fiber bundles were used for background correction in all analyses. ESR1 mRNA abundance per kisspeptin neuron In the POA/PeV and ARC, the number of kisspeptin neurons was determined. Four sections within the POA (including two sections at the level of the OVLT), three through the rARC, four through the mARC, and two through the cARC were selected. Bright-field images were captured at ×40 magnification from 20 kisspeptin neurons in at least three representative sections of the POA, 15 kisspeptin neurons in at least three representative sections of the rARC, 30 kisspeptin neurons in at least four representative sections of the mARC, and 15 kisspeptin neurons in at least two representative sections of the cARC of each ewe. A threshold signal was established and applied to all images and an ROI of 20 µm in diameter was placed over a kisspeptin neuron identified in the image. The number of objects representing the area covered by silver grains was determined in the standardized ROI and recorded. Fifteen ROIs of 20 µm in diameter were placed randomly in an image captured from a neuronal fiber bundle present in the section (i.e., anterior commissure and fornix) to determine background number of silver grains in each section. The average number of silver grains determined in these 15 ROIs was used to adjust the data obtained from images of each neuron. Kisspeptin neurons containing fivefold or more the number of silver grains determined in the background image were identified as ESR1-expressing kisspeptin neurons. ESR1 content per kisspeptin neuron To determine the percentage of kisspeptin neurons in the POA and ARC that contained ESR1, a virtual slide microscope (VS120; Olympus, Tokyo, Japan) was used. After all sections were scanned and digitized using the ×20 objective, four sections within the POA (including two sections at the level of the OVLT), three through the rARC, four through the mARC, and two through the cARC were analyzed using OlyVIA software from Olympus to determine the percentage of kisspeptin neurons that contained ESR1 protein. Hormone assays Concentrations of LH in plasma samples were determined by a double antibody radioimmunoassay previously validated and reported from this laboratory (20). Intra-assay and interassay coefficients of variation were 11.60% and 19.96%, respectively. Statistical analysis Frequency and amplitude of LH pulses were determined using the Pulse4 pulse-detection algorithm available within the Pulse XP program (21). Adjusted ESR1 expression data were transformed to the log10, and the percentage of kisspeptin neurons expressing ESR1 was transformed using the arcsine square root method. Normalized ESR1 expression, the number of kisspeptin neurons, the transformed percentage of kisspeptin neurons expressing ESR1, the mean adjusted number of silver grains per kisspeptin neuron, and the percentage of kisspeptin neurons that contained ESR1 were analyzed by a t test (JM Pro 12; SAS Institute, Cary, NC). Mean concentrations of LH and the amplitude of LH pulses were analyzed using a mixed model analysis for repeated measures (Proc Mixed; SAS Institute) to assess main effects (treatment) and the treatment by time interaction. Ewe ID was the random effect, time was the repeated variable, and ewe ID was the subject. The frequency of LH pulses between OVX and OVX+E lambs was compared using a Wilcoxon–Mann–Whitney test. Means for LH variables are reported as least squares means [± standard error of the mean (SEM)]. Because the frequency of LH pulses was highly variable among ewe lambs in the OVX+E group, lambs were reassigned to one of three separate groups based on the number of LH pulses detected in 12 hours as follows: (1) low frequency (LF; 1 or 2 pulses per 12 hours; n = 3), (2) moderate frequency (MF; 6 or 7 pulses per 12 hours; n = 6), and (3) high frequency (HF; 10 or more pulses per 12 hours; n = 5) and post hoc analyses were performed. Mean concentrations of LH and pulse amplitude data were reanalyzed using Proc Mixed for repeated measures, with main effects of group (OVX, LF, MF, and HF) and the group by time interaction. When significant differences were observed, a Tukey post hoc test was used to compare means. The frequency of LH pulses among the groups was compared as described above using a Wilcoxon–Mann–Whitney test. All other tissue-related data were also reanalyzed based on pulse frequency category using a one-way analysis of variance (ANOVA). The main source of variation was group (OVX, LF, MF, and HF). When significant differences were observed in the ANOVA, a Tukey post hoc test was used to compare means between groups. Results Mean (±SEM) body weight of ewe lambs at 30 weeks of age was 50 ± 1.5 kg and did not differ between OVX and OVX+E ewe lambs. Mean circulating concentrations of LH were greater (P < 0.0001) in OVX (5.5 ± 0.5 ng/mL) than in OVX+E lambs (2.2 ± 0.4 ng/mL). This was accompanied by a greater overall frequency (14.6 ± 1.6 vs 7.8 ± 1.2 pulses per12 hours (P < 0.001) of LH pulses in OVX than in OVX+E lambs with no differences in LH pulse amplitude between the groups (4.0 ± 0.5 ng/mL vs 3.0 ± 0.5 ng/mL). However, lambs in the OVX+E group exhibited significant variability in the frequency of LH pulses, ranging from 1 to 20 pulses per 12 hours. Therefore, in a post hoc analysis, the OVX+E group was repartitioned into three groups based on the number of LH pulses as follows: (1) LF (1 to 2 pulses per 12 hours; n = 3), (2) MF (6 to 7 pulses per 12 hours; n = 6), and (3) HF (10 to 20 pulses per 12 hours; n = 5). Patterns of LH release during the 12-hour sampling period from one representative lamb in each group are presented in Fig. 1A. Analysis indicated that the frequency of LH pulses in OVX and OVX+E HF lambs was similar, both of which were greater (P < 0.0001) than in MF and LF groups, which also differed (Fig. 1B). Differences in concentrations of LH favored OVX over all other groups, and concentrations of LH in HF were greater than LF (P < 0.05; Fig. 1B). There were no differences in LH pulse amplitude between any of the groups. Figure 1. View largeDownload slide (A) Patterns of LH release in representative OVX and OVX+E lambs receiving an estradiol implant exhibiting HF, MF, or LF release. Detected pulses are indicated with asterisks. (B) Least squares mean (±SEM) concentrations of plasma LH and frequency and amplitude of LH pulses in OVX lambs and in OVX+E lambs exhibiting HF, MF, or LF of LH pulses. Mean LH concentrations and amplitude of LH pulses were analyzed using a mixed model analysis for repeated measures; frequency of LH pulses was compared using a Wilcoxon–Mann–Whitney test. Means with different superscripts differ (P < 0.05). Figure 1. View largeDownload slide (A) Patterns of LH release in representative OVX and OVX+E lambs receiving an estradiol implant exhibiting HF, MF, or LF release. Detected pulses are indicated with asterisks. (B) Least squares mean (±SEM) concentrations of plasma LH and frequency and amplitude of LH pulses in OVX lambs and in OVX+E lambs exhibiting HF, MF, or LF of LH pulses. Mean LH concentrations and amplitude of LH pulses were analyzed using a mixed model analysis for repeated measures; frequency of LH pulses was compared using a Wilcoxon–Mann–Whitney test. Means with different superscripts differ (P < 0.05). Overall ESR1 mRNA abundance in the POA and hypothalamus Accumulation of silver grains following in situ hybridization for detection of ESR1 mRNA was observed throughout the POA and hypothalamus (Fig. 2). There were no differences between OVX and OVX+E lambs in the number of silver grains per ROI in the POA (937 ± 97), PeV (724 ± 53), SON (1253 ± 112), PVN (689 ± 72), VMH (559 ± 61), VL-VMH (829 ± 83), and cARC (697 ± 114). In the mARC, OVX lambs exhibited greater ESR1 mRNA abundance (P < 0.05) than did OVX+E lambs, albeit this difference was small (Fig. 2C). There was also a trend (P < 0.08) for OVX lambs to exhibit greater ESR1 mRNA abundance in the rARC than OVX+E lambs (Fig. 2C). No differences were observed among HF, MF, and LF OVX+E lambs in any of the areas studied. Figure 2. View largeDownload slide (A) Detection of ESR1 mRNA in the hypothalamus. Drawing of a hypothalamic section containing the VL-VMH and mARC. (B) Low-magnification image of the boxed area in (A) depicting signal at the level of the mARC and a portion of the VL-VMH. (C) Normalized mean (±SEM) number of silver grains in the rostral (rARC, top panel), middle (mARC, middle panel) and caudal (cARC, bottom panel) portions of the ARC in OVX and OVX+E lambs. The number of silver grains in the rARC (top panel) of OVX lambs tended to be greater (t test; #P < 0.08) than in the OVX+E lambs. The number of silver grains in the mARC (middle panel) of OVX lambs was greater (t test; *P < 0.05) than in OVX+E lambs. No differences were observed in the cARC (bottom panel). The asterisk in (B) indicates an artifact associated with meninges. 3V, third ventricle; fx, fornix; pt, pars tuberalis. Figure 2. View largeDownload slide (A) Detection of ESR1 mRNA in the hypothalamus. Drawing of a hypothalamic section containing the VL-VMH and mARC. (B) Low-magnification image of the boxed area in (A) depicting signal at the level of the mARC and a portion of the VL-VMH. (C) Normalized mean (±SEM) number of silver grains in the rostral (rARC, top panel), middle (mARC, middle panel) and caudal (cARC, bottom panel) portions of the ARC in OVX and OVX+E lambs. The number of silver grains in the rARC (top panel) of OVX lambs tended to be greater (t test; #P < 0.08) than in the OVX+E lambs. The number of silver grains in the mARC (middle panel) of OVX lambs was greater (t test; *P < 0.05) than in OVX+E lambs. No differences were observed in the cARC (bottom panel). The asterisk in (B) indicates an artifact associated with meninges. 3V, third ventricle; fx, fornix; pt, pars tuberalis. Number and distribution of kisspeptin neurons in the POA and ARC The number of kisspeptin-immunoreactive neurons detected in the POA of OVX lambs was limited, with no or only a few (up to four) neurons observed in each lamb. Therefore, no comparisons between OVX and OVX+E lambs were performed for the POA. In contrast to OVX lambs, numerous kisspeptin neurons were observed in the POA of OVX+E lambs (257 ± 37). However, no differences in the number of kisspeptin-immunoreactive neurons in the POA were observed among OVX+E lambs in HF, MF, and LF groups. Kisspeptin-immunoreactive neurons were readily detected in the ARC (Fig. 3A) in substantial numbers in both OVX and OVX+E lambs. The number of kisspeptin neurons was greater (P < 0.001) in OVX than in OVX+E lambs in the rARC and mARC (Fig. 4). In the cARC, the number of kisspeptin neurons was also greater in OVX than OVX+E lambs, but the difference was not statistically significant (P < 0.11; Fig. 4C). There were no differences among OVX+E lambs in HF, MF, and LF groups for the number of kisspeptin-immunoreactive neurons in any of the three subdivisions of the ARC. Figure 3. View largeDownload slide Images of a section at the level of the ARC processed for dual-label detection of ESR1-containing and kisspeptin-immunoreactive neurons. (A) Low-magnification image depicting kisspeptin-immunoreactive neurons (brown) and cells stained nonspecifically with cresyl violet (purple) in the mARC. (B) High-magnification image of two kisspeptin neurons shown in (A) (arrows) exhibiting silver grain accumulation over the brown-stained cell body and proximal dendrite. (C) High-magnification image of a kisspeptin neuron shown in (A) (arrowhead) exhibiting only few silver grains accumulated over the brown-stained cell body and proximal dendrites. 3V, third ventricle. Figure 3. View largeDownload slide Images of a section at the level of the ARC processed for dual-label detection of ESR1-containing and kisspeptin-immunoreactive neurons. (A) Low-magnification image depicting kisspeptin-immunoreactive neurons (brown) and cells stained nonspecifically with cresyl violet (purple) in the mARC. (B) High-magnification image of two kisspeptin neurons shown in (A) (arrows) exhibiting silver grain accumulation over the brown-stained cell body and proximal dendrite. (C) High-magnification image of a kisspeptin neuron shown in (A) (arrowhead) exhibiting only few silver grains accumulated over the brown-stained cell body and proximal dendrites. 3V, third ventricle. Figure 4. View largeDownload slide Mean (±SEM) number of kisspeptin-immunoreactive neurons in the (A) rARC, (B) mARC, and (C) cARC. The mean number of kisspeptin neurons was greater (t test; **P < 0.001) in the rARC and mARC of OVX lambs than in OVX+E lambs. Figure 4. View largeDownload slide Mean (±SEM) number of kisspeptin-immunoreactive neurons in the (A) rARC, (B) mARC, and (C) cARC. The mean number of kisspeptin neurons was greater (t test; **P < 0.001) in the rARC and mARC of OVX lambs than in OVX+E lambs. ESR1 mRNA abundance in kisspeptin neurons A considerable number of kisspeptin neurons of both POA and ARC populations were observed to contain ESR1 (Fig. 3B), although many were also observed to exhibit no meaningful accumulation of silver grains (Fig. 3C). In the POA of OVX+E lambs, the percentage of ESR1-positive kisspeptin neurons was 58% ± 7%, and the percentage of ESR1-positive kisspeptin neurons did not differ among OVX+E lambs in the HF, MF, and LF groups. The mean number of silver grains per ESR1-positive kisspeptin neuron in the POA of OVX+E lambs also did not differ among the HF, MF, and LF groups (9.4 ± 0.5). In the ARC, the percentage of ESR1-positive kisspeptin neurons was 50%, 44%, and 37% for the rARC, mARC, and cARC, respectively, and did not differ between OVX and OVX+E lambs. However, the percentage of ESR1-positive neurons in the mARC was greater (P < 0.05) in HF than in LF lambs (Fig. 5) and tended to be greater (P < 0.06) than the MF group (Fig. 5). The mean percentage of ESR1-positive kisspeptin neurons located in the mARC in OVX lambs did not differ from HF and MF groups but tended to be greater (P < 0.06) than in the LF group (Fig. 5). The mean number of silver grains per kisspeptin neuron also tended to be greater (P < 0.07) in the rARC and mARC of OVX than in OVX+E lambs, but did not differ in the cARC (Fig. 6). When comparing HF, MF, and LF groups, there were no differences in the mean number of silver grains per kisspeptin neuron. Figure 5. View largeDownload slide Mean (±SEM) percentage of ESR1-positive kisspeptin neurons in the mARC of OVX lambs and OVX+E lambs exhibiting HF, MF, and LF of LH pulses (one-way ANOVA; #P < 0.06, *P < 0.05). Figure 5. View largeDownload slide Mean (±SEM) percentage of ESR1-positive kisspeptin neurons in the mARC of OVX lambs and OVX+E lambs exhibiting HF, MF, and LF of LH pulses (one-way ANOVA; #P < 0.06, *P < 0.05). Figure 6. View largeDownload slide Mean (±SEM) number of grains per kisspeptin neuron in OVX and OVX+E lambs in the (A) rARC, (B) mARC, and (C) cARC. The average number of grains per kisspeptin neuron tended (t test; #P < 0.07) to be greater in OVX lambs than in OVX+E lambs in the rARC and mARC. Figure 6. View largeDownload slide Mean (±SEM) number of grains per kisspeptin neuron in OVX and OVX+E lambs in the (A) rARC, (B) mARC, and (C) cARC. The average number of grains per kisspeptin neuron tended (t test; #P < 0.07) to be greater in OVX lambs than in OVX+E lambs in the rARC and mARC. Colocalization of kisspeptin neurons with ESR1 protein A high percentage of kisspeptin neurons in both the POA and ARC were found to contain ESR1 (Fig. 7). In the POA of OVX+E lambs, 90% ± 2% of kisspeptin neurons were found to contain ESR1 but did not differ among OVX+E HF, MF, and LF groups. Whereas a high percentage of kisspeptin neurons in the ARC of both OVX and OVX+E lambs contained ESR1 (82.6% ± 1.3%), a greater percentage (P < 0.05) of kisspeptin neurons in OVX lambs contained ESR1 compared with OVX+E lambs in the rARC, mARC, and cARC (Fig. 8). The mean percentage of kisspeptin neurons that contained ESR1 did not differ among OVX+E HF, MF, and LF groups. Figure 7. View largeDownload slide High-magnification image depicting kisspeptin-immunoreactive neurons (brown) and ESR1 (black) in the mARC. Representative kisspeptin neurons that contain ESR1 are indicated by white arrows, and representative kisspeptin neurons that do not contain ESR1 are indicated by black arrows. Figure 7. View largeDownload slide High-magnification image depicting kisspeptin-immunoreactive neurons (brown) and ESR1 (black) in the mARC. Representative kisspeptin neurons that contain ESR1 are indicated by white arrows, and representative kisspeptin neurons that do not contain ESR1 are indicated by black arrows. Figure 8. View largeDownload slide Mean (±SEM) percentage of kisspeptin neurons containing ESR1 in OVX and OVX+E lambs in the (A) rARC, (B) mARC, and (C) cARC. The percentage of kisspeptin neurons that contained ESR1 was greater in OVX than in OVX+E lambs in the rARC (t test; *P < 0.05), mARC (t test; **P < 0.001), and cARC (t test; *P < 0.05). Figure 8. View largeDownload slide Mean (±SEM) percentage of kisspeptin neurons containing ESR1 in OVX and OVX+E lambs in the (A) rARC, (B) mARC, and (C) cARC. The percentage of kisspeptin neurons that contained ESR1 was greater in OVX than in OVX+E lambs in the rARC (t test; *P < 0.05), mARC (t test; **P < 0.001), and cARC (t test; *P < 0.05). Discussion The results of experiments reported in this study indicate that, although estradiol reduces the number of kisspeptin neurons in prepubertal/peripubertal ewe lambs, the escape from estradiol negative feedback, although clearly a fundamental element of pubertal maturation in sheep (2, 8, 22, 23), is not associated with declines in ESR1 expression or ESR1 protein in kisspeptin neurons. Indeed, as the escape from estradiol negative feedback became evident in HF and MF females in the present experiment, ESR1 expression in kisspeptin neurons increased relative to LF lambs and did not differ from OVX only females. Similarly, abundant ESR1 protein in kisspeptin neurons was observed in all groups, with the greatest amount observed in OVX lambs. Studies in mice have indicated that ESR1 is the major type of estrogen receptor mediating estradiol negative feedback effects on GnRH secretion (9), and neurons located in the ARC that contain ESR1 are essential for communicating estradiol negative feedback (24). In a study using intact heifers, a reduction in the overall number of estradiol receptors in the anterior and mediobasal hypothalamus was observed to occur as heifers approached puberty (25). A similar decline in the number of estrogen receptors in the hypothalamus was also detected during juvenile development in female rats (26). Because both studies used estradiol-binding assays of hypothalamic extracts, the specific type of estrogen receptor investigated, as well as the regional location within the hypothalamus in which receptors were present, was not determined. Therefore, a direct comparison of those data with changes in overall ESR1 mRNA abundance observed in the present study should be done with caution. Nevertheless, the results of the present study in ewe lambs suggest that the decrease in estradiol binding in the hypothalamus reported during reproductive maturation in rats (26) and heifers (25) are not negatively associated with changes in overall ESR1 mRNA abundance or ESR1 protein. The effects of estradiol on the regulation of ESR1 mRNA abundance in specific hypothalamic areas appear more consistent among the various studies reported. Estrogen receptor mRNA in the VMH and ARC decreases with estradiol treatment in mature, ovariectomized rats (27). Although we did not observe changes in ESR1 mRNA in the VMH between OVX and OVX+E lambs, estradiol treatment decreased the overall abundance of ESR1 mRNA in the ARC. Whether the difference in ESR1 expression in the VMH reflects distinction among species, or represents differences in the animal model as related to the maturation of neuroendocrine function, is unclear. Nevertheless, the consistency between studies on the effects of estradiol in the regulation of ESR1 mRNA abundance in the ARC indicates the relevance of this hypothalamic area for estradiol’s control of reproductive function in females. The presence or absence of estradiol has also been shown to affect ESR1 immunoreactivity. In rats, estradiol downregulates estrogen receptor immunoreactivity in the medial preoptic nucleus (28). The density of ESR1-immunoreactive neurons was lower in the AVPV, VL-VMH, and ARC of ovariectomized rats treated with estradiol benzoate than in untreated rats (29). Abundance of Kiss1 mRNA has also been demonstrated to be influenced by estradiol. In mature ewes, Kiss1 expression in the ARC is increased owing to ovariectomy, and estradiol replacement decreases Kiss1 mRNA in the ARC to levels similar to those of intact ewes (30). Likewise, ovariectomy increases the number of immunoreactive kisspeptin neurons in the ARC (31). In the present study, we observed that the number of kisspeptin neurons in the rARC and mARC was greater in ovariectomized ewe lambs that did not receive an estradiol implant. This observation is consistent with the findings of the study reported by Nestor et al. (32) in which the number of kisspeptin neurons increased in the ARC of prepubertal ewes that were ovariectomized compared with intact ewes. The study by Nestor et al. (32) also reported that the number of kisspeptin neurons was greater in postpubertal compared with prepubertal intact ewes. Because the frequency of LH pulses increases as ewes approach maturity (33), it was plausible to expect that the number of kisspeptin neurons would increase in the ARC as the frequency of LH pulses increased in peripubertal lambs of the present study. An earlier study in our laboratory indicating that Kiss1 mRNA abundance increases in the ARC in association with increased LH pulsatility in ovariectomized, estradiol-treated lambs (15) supports this hypothesis. However, in the present study, no differences in the number of kisspeptin neurons were detected in the ARC among groups of ovariectomized, estradiol-implanted lambs exhibiting high, moderate, or low LH pulsatility. Thus, immediate changes in kisspeptin synthesis and accumulation in ARC neurons do not appear to clearly precede the decrease in the sensitivity to estradiol negative feedback during reproductive maturation in sheep. Estradiol likely regulates Kiss1 mRNA abundance directly because ESR1 is detected in kisspeptin neurons in sheep (11). Therefore, it has been proposed that kisspeptin neurons mediate the estradiol feedback regulation of GnRH and LH secretion. Research in mice has demonstrated that Esr1 in kisspeptin neurons is critical for the control of LH pulsatility and reproductive function. Selective ablation of Esr1 in kisspeptin neurons in female mice increases Kiss1 mRNA in the ARC and circulating concentrations of LH (13). Additionally, whereas these mice exhibit increased concentrations of LH and advanced vaginal opening, normal estrous cyclicity is absent. The ability of estradiol to suppress LH secretion and Kiss1 mRNA abundance in prepubertal but not adult female mice is also dependent on the presence of Esr1 in kisspeptin neurons (14, 34). These observations support the role for Esr1 in kisspeptin neurons as important for signaling estradiol feedback control of gonadotropin release in mice (13, 14). Therefore, even though only small differences in overall ESR1 expression in the ARC between OVX and OVX+E prepubertal ewes were observed in this study, it is seemingly plausible to expect such changes in a neuronal population as critically important to reproduction (e.g., kisspeptin neurons). We hypothesized that downregulation of ESR1 in kisspeptin neurons could facilitate the increased kisspeptin synthesis and release during the pubertal transition and downstream stimulation of GnRH secretory activity. However, contrary to our hypothesis, we observed that the percentage of kisspeptin neurons in the ARC expressing ESR1 mRNA was greater in OVX+E lambs exhibiting a high frequency of LH pulses than in less reproductively mature lambs. Thus, the ability of estradiol to inhibit ESR1 transcription in kisspeptin neurons in the ARC appears less in lambs exhibiting a more advanced stage of reproductive maturation and decreased sensitivity to estradiol negative feedback. Because the percentage of kisspeptin neurons in the ARC that expressed ESR1 mRNA increased instead of decreased in OVX+E lambs that exhibited increased LH pulsatility, we also examined the percentage of kisspeptin neurons that contained ESR1 protein in the ARC of OVX and OVX+E lambs. Regardless of treatment group, we found that a greater percentage of kisspeptin neurons (>90%) contained ESR1 protein compared with ESR1 mRNA (50%). These differences are likely due to the different thresholds of detection between the techniques of in situ hybridization and immunocytochemistry. In keeping with previous reports from this laboratory (15, 35), kisspeptin neurons in the present experiment were required to contain fivefold more silver grains than the background signal to be considered as expressing ESR1 mRNA. This is a relatively stringent threshold. If this threshold is decreased to threefold the background, the percentage of kisspeptin cells that are also considered positive for ESR1 mRNA increases to ∼85%, comparable to that estimated for containing ESR1 protein. The percentage of kisspeptin neurons that contained ESR1 protein remained slightly but significantly greater in OVX lambs compared with OVX+E lambs, and there were no differences among HF, MF, and LF groups. Because the presence of estradiol is known to decrease the number of kisspeptin neurons in the ARC of prepubertal ewe lambs (32), the difference observed in the percentage of kisspeptin neurons that contain ESR1 for OVX and OVX+E ewes may be due to a decrease in the number of kisspeptin neurons. Therefore, although estradiol decreases the percentage of kisspeptin neurons that contain ESR1 protein in the ARC of lambs, other mechanisms appear to be more directly involved in controlling the escape from estradiol negative feedback. It is also possible that the number of ESR1 receptors present within an individual kisspeptin neuron is changing as LH pulse frequency increases. However, the use of immunocytochemistry to detect kisspeptin and ESR1 protein does not provide a strategy for answering this question. Immunocytochemistry is a semiquantitative technique whereby cells are classified as either expressing or not expressing a protein. A more quantitative technique would be needed to answer this question. In addition to participating in the communication of estradiol negative feedback to GnRH neurons, kisspeptin neurons are likely to also be involved in the transmission of estradiol positive feedback. In rodents, kisspeptin neurons located in the POA appear to play such a role (36–40). However, the location of kisspeptin neurons contributing to estradiol positive feedback in sheep is more ambiguous than in rodents. Whereas the expression of KISS1 in the POA increases during the late follicular phase in ewes (41), as well as during juvenile development (11, 15, 41), the percentage of kisspeptin neurons in both the POA and ARC exhibiting Fos activity (a marker of neuronal activation) is observed to increase during the preovulatory surge of LH in mature ewes (42). In contrast, Hoffman et al. (43) observed Fos labeling only in kisspeptin neurons in the POA during the preovulatory surge in ewes. Therefore, the role of kisspeptin neurons in the ARC in mediating estradiol stimulation of the preovulatory LH surge in the ewe requires further elucidation. In the absence of estradiol implants, few kisspeptin neurons were observed in the POA of OVX lambs. This is in agreement with previous reports in sheep (32) and mice (13, 44). The previous observation that the numbers of kisspeptin neurons do not change as the frequency of LH pulses increases in 30-week-old ewe lambs is also in agreement with our previous study (15). Although estradiol is required for activation of KISS1 mRNA in the POA, estradiol does not appear to regulate ESR1 mRNA abundance in kisspeptin neurons in the POA in a manner similar to that in the ARC. However, the percentage of kisspeptin neurons that contained ESR1 protein was high in both the POA and ARC. A much greater percentage of kisspeptin neurons in the POA was found to contain ESR1 in this study when compared with a previous study conducted in adult ewes in the luteal phase (11). It is possible that these discrepancies were caused by differences in endocrine status or age of the animals used in the two studies. To our knowledge, no studies have been conducted to determine whether the percentage of kisspeptin neurons that contain ESR1 changes throughout the estrous cycle in female sheep. In summary, contrary to our hypothesis, the increase in LH pulsatility in estradiol-treated, maturing ewe lambs was associated with enhanced ESR1 mRNA abundance in kisspeptin neurons in the ARC, and the absence of estradiol in OVX ewe lambs was associated with the greatest ESR1 abundance and percentage of kisspeptin neurons containing ESR1 protein in the ARC. Therefore, changes in the expression of ESR1, particularly in kisspeptin neurons in the ARC, fail to explain the pubertal escape from estradiol negative feedback in ewe lambs. Appendix. Antibody Table Peptide/Protein Target  Antigen Sequence  Name of Antibody  Manufacturer, Catalog No, and/or Name of Individual Providing the Antibody  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  Kisspeptin  Peptide from mouse kisspeptin 10  Anti-kisspeptin antibody  Gifted by A. Caraty  Rabbit; polyclonal  1:75,000  AB_2622231  Kisspeptin  Peptide from mouse kisspeptin 10  Anti-kisspeptin antibody  Millipore, AB9754  Rabbit; polyclonal  1:2000  AB_229652  ERα  Recombinant human estrogen receptor protein  ERα (clone 1D5)  Dako, M7047  Mouse; monoclonal  0.388888889  AB_2101946  LH  Ovine LH  Anti-oLH ab  A.F. Parlow, National Pituitary Hormone Program  Rabbit  1:125,000  AB_2716713  Peptide/Protein Target  Antigen Sequence  Name of Antibody  Manufacturer, Catalog No, and/or Name of Individual Providing the Antibody  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID  Kisspeptin  Peptide from mouse kisspeptin 10  Anti-kisspeptin antibody  Gifted by A. Caraty  Rabbit; polyclonal  1:75,000  AB_2622231  Kisspeptin  Peptide from mouse kisspeptin 10  Anti-kisspeptin antibody  Millipore, AB9754  Rabbit; polyclonal  1:2000  AB_229652  ERα  Recombinant human estrogen receptor protein  ERα (clone 1D5)  Dako, M7047  Mouse; monoclonal  0.388888889  AB_2101946  LH  Ovine LH  Anti-oLH ab  A.F. Parlow, National Pituitary Hormone Program  Rabbit  1:125,000  AB_2716713  View Large Abbreviations: ANOVA analysis of variance ARC arcuate nucleus cARC caudal ARC cDNA complementary DNA DAB 3,3′-diaminobenzidine ESR1 estrogen receptor α HF high frequency LF low frequency LH luteinizing hormone mARC middle ARC MF moderate frequency mRNA messenger RNA NGS normal goat serum OVLT organum vasculosum of the lamina terminalis OVX ovariectomy without estradiol implant OVX+E ovariectomy with estradiol implant PBS phosphate-buffered saline PBSTX phosphate-buffered saline containing 0.4% Triton X-100 PCR polymerase chain reaction PeV periventricular nucleus POA preoptic area PVN paraventricular nucleus rARC rostral ARC ROI region of interest RRID Research Resource Identifier SEM standard error of the mean SON supraoptic nucleus SSC saline sodium citrate VL-VMH ventrolateral–ventromedial hypothalamus VMH ventromedial hypothalamus. 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Published: Jan 1, 2018

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