Chronic estradiol exposure suppresses luteinizing hormone surge without affecting kisspeptin neurons and estrogen receptor alpha in anteroventral periventricular nucleus†.

Mammalian ovulation is induced by a luteinizing hormone surge, which is triggered by elevated plasma estrogen levels; however, chronic exposure to high levels of estradiol is known to inhibit luteinizing hormone secretion. In the present study, we hypothesized that the inhibition of the luteinizing hormone surge by chronic estradiol exposure is due to the downregulation of the estrogen receptor alpha in kisspeptin neurons at hypothalamic anteroventral periventricular nucleus, which is known as the gonadotropin-releasing hormone/luteinizing hormone surge generator. Animals exposed to estradiol for 2 days showed an luteinizing hormone surge, whereas those exposed for 14 days showed a significant suppression of luteinizing hormone. Chronic estradiol exposure did not affect the number of kisspeptin neurons and the percentage of kisspeptin neurons with estrogen receptor alpha or c-Fos in anteroventral periventricular nucleus, but it did affect the number of kisspeptin neurons in arcuate nucleus. Furthermore, chronic estradiol exposure did not affect gonadotropin-releasing hormone neurons. In the pituitary, 14-day estradiol exposure significantly reduced the expression of Lhb mRNA and LHß-immunoreactive areas. Gonadotropin-releasing hormone-induced luteinizing hormone release was also reduced significantly by 14-day estradiol exposure. We revealed that the suppression of an luteinizing hormone surge by chronic estradiol exposure was induced in association with the significant reduction in kisspeptin neurons in arcuate nucleus, luteinizing hormone expression in the pituitary, and pituitary responsiveness to gonadotropin-releasing hormone, and this was not caused by changes in the estrogen receptor alpha-expressing kisspeptin neurons in anteroventral periventricular nucleus and gonadotropin-releasing hormone neurons, which are responsible for estradiol positive feedback.

Transcriptomic Profiling of Lactotroph Pituitary Neuroendocrine Tumors via RNA Sequencing and Ingenuity Pathway Analysis.

INTRODUCTION: Lactotroph pituitary neuroendocrine tumors (PitNETs) are common pituitary tumors, but their underlying molecular mechanisms remain unclear. This study aimed to investigate the transcriptomic landscape of lactotroph PitNETs and identify potential molecular mechanisms and therapeutic targets through RNA sequencing and ingenuity pathway analysis (IPA). METHODS: Lactotroph PitNET tissues from five surgical cases without dopamine agonist treatment underwent RNA sequencing. Normal pituitary tissues from 3 patients served as controls. Differentially expressed genes (DEGs) were identified, and the functional pathways and gene networks were explored by IPA. RESULTS: Transcriptome analysis revealed that lactotroph PitNETs had gene expression patterns that were distinct from normal pituitary tissues. We identified 1,172 upregulated DEGs, including nine long intergenic noncoding RNAs (lincRNAs) belonging to the top 30 DEGs. IPA of the upregulated DEGs showed that the estrogen receptor signaling, oxidative phosphorylation signaling, and EIF signaling were activated. In gene network analysis, key upstream regulators, such as EGR1, PRKACA, PITX2, CREB1, and JUND, may play critical roles in lactotroph PitNETs. CONCLUSION: This study provides a comprehensive transcriptomic profile of lactotroph PitNETs and highlights the potential involvement of lincRNAs and specific signaling pathways in tumor pathogenesis. The identified upstream regulators may be potential therapeutic targets for future investigations.

Effects of general anesthesia on behavioral circadian rhythms and clock-gene expression in the suprachiasmatic nucleus in rats.

The suprachiasmatic nucleus (SCN) of the hypothalamus is a nucleus that regulates circadian rhythms through the cyclic expression of clock genes. It has been suggested that circadian-rhythm-related, adverse postoperative events, including sleep disturbances and delirium, are partly caused by anesthesia-induced disruption of clock-gene expression. We examined the effects of multiple general anesthetics on the expression cycle of Period2 (Per2), one of the clock genes that regulate circadian rhythms in the SCN, and on the behavioral rhythms of animals. Rats were treated with sevoflurane, propofol, and dexmedetomidine for 4 h. The expression of Per2 in SCN was analyzed using in situ hybridization, and the behavioral rhythm before and after anesthesia was analyzed. Per2 expression in the SCN decreased significantly immediately after anesthesia in all groups compared with corresponding control groups. However, Per2 returned to normal levels within 24 h, and there was no phase change in the gene expression cycle or behavioral rhythm. This study suggests that acute suppression of Per2 expression may be a general phenomenon induced by general anesthesia, but that the molecular mechanism of the body clock is resilient to disturbances to some extent.

Kisspeptin neurons as an integration center of reproductive regulation: Observation of reproductive function based on a new concept of reproductive regulatory nervous system.

BACKGROUND: Regulation of the reproductive system has been explained by the actions and feedback of gonadotropin releasing hormone-luteinizing hormone/follicle stimulating hormone (GnRH-LH/FSH) -sex steroids; however, the discovery of kisspeptin neurons and a kisspeptin-GnRH-LH/FSH axis has prompted this regulation to be reviewed. METHODS: We investigated changes in kisspeptin neurons and associated changes in the hypothalamic-pituitary-gonadal (HPG) axis under various situations and experimental conditions using histochemical methods. MAIN FINDINGS RESULTS: Kisspeptin neurons play an important role in receiving and integrating information from internal and external environmental factors and communicating it to the conventional HPG axis. CONCLUSION: The recently described Kisspeptin-GnRH-LH/FSH-gonad system regulates reproductive function via mechanisms that until recently were not completely understood.

Expression analysis of neuropeptide FF receptors on neuroendocrine-related neurons in the rat brain using highly sensitive in situ hybridization.

RF-amide peptides, a family of peptides characterized by a common carboxy-terminal Arg-Phe-NH2 motif, play various physiological roles in the brain including the modulation of neuroendocrine signaling. Neuropeptide FF (NPFF) receptors exhibit a high affinity for all RF-amide peptides, which suggests that the neurons expressing these NPFF receptors may have multiple functions in the brain. However, the distribution of the neurons expressing NPFF receptors in the rat brain remains poorly understood. This study aimed to determine the detailed histological distribution of mRNA that encodes the neuropeptide FF receptors (Npffr1 and Npffr2) in the rat brain using in situ hybridization. Neurons with strong Npffr1 expression were observed in the lateral septal nucleus and several hypothalamic areas related to neuroendocrine functions, including the paraventricular nucleus (PVN) and arcuate nucleus, whereas Npffr2-expressing neurons were observed mainly in brain regions involved in somatosensory pathways, such as several subnuclei of the thalamus. Npffr1 expression was observed in 70% of corticotropin-releasing hormone neurons, but in only a small population of oxytocin and vasopressin neurons in the PVN. Npffr1 expression was also observed in the dopaminergic neurons in the periventricular nucleus and the dorsal arcuate nucleus, and in the kisspeptin neurons in the anteroventral periventricular nucleus. These results suggest that NPFFR1-mediated signaling may be involved in neuroendocrine functions, such as in reproduction and stress response. In conjunction with a detailed histological map of NPFFRs, this study provides useful data for future neuroendocrine research.

Comprehensive Dipeptide Profiling and Quantitation by Capillary Electrophoresis and Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Dipeptides have attracted much attention as post-amino acids with physical properties and functions different from those of amino acids. However, a given dipeptide cannot be distinguished by mass spectrometry from its structural isomer with an opposite amino acid binding order unless these isomers are separated before introduction, which complicates the comprehensive analysis of dipeptides. Herein, a novel analytical platform for dipeptide analysis by capillary electrophoresis tandem mass spectrometry and liquid chromatography tandem mass spectrometry is developed. This method is used to quantitate 335 dipeptides and achieves excellent separation of structural isomers with opposite binding orders, high correlation coefficients, and low instrumental detection limits (0.088-83.1 nM). Moreover, acceptable recoveries (70-135%) are observed for most tested dipeptides in chicken liver samples spiked both before and after preparation. The developed method is also applied to the quantitation of dipeptides in the livers of mice fed different diets to detect 236 dipeptides, and the shift from a normal diet to a high-fat diet is shown to increase/decrease (p < 0.05, fold-change < 0.5) the contents of 0/29 dipeptides, respectively. The developed method is expected to facilitate the search for new dipeptide applications such as novel functional components of foods and biomarkers of diseases.

Mating-induced increase in Kiss1 mRNA expression in the anteroventral periventricular nucleus prior to an increase in LH and testosterone release in male rats.

Kisspeptin has an indispensable role in gonadotropin-releasing hormone/gonadotropin secretion in mammals. In rodents, kisspeptin neurons are located in distinct brain regions, namely the anteroventral periventricular nucleus-periventricular nucleus continuum (AVPV/PeN), arcuate nucleus (ARC), and medial amygdala (MeA). Among them, the physiological role of AVPV/PeN kisspeptin neurons in males has not been clarified yet. The present study aims to investigate the acute effects of the olfactory and/or mating stimulus with a female rat on hypothalamic and MeA Kiss1 mRNA expression, plasma luteinizing hormone (LH) and testosterone levels in male rats. Intact male rats were exposed to the following stimuli: exposure to clean bedding; exposure to female-soiled bedding as a female-olfactory stimulus; exposure to female-soiled bedding and mating stimulus with a female rat. The mating stimulus significantly increased the number of the AVPV/PeN Kiss1 mRNA-expressing cells in males within 5 minutes after the exposure, and significantly increased LH and testosterone levels, followed by an increase in male sexual behavior. Whereas, the males exposed to female-soiled bedding showed a moderate increase in LH levels and no significant change in testosterone levels and the number of the AVPV/PeN Kiss1 mRNA-expressing cells. Importantly, none of the stimuli affected the number of Kiss1 mRNA-expressing cells in the ARC and MeA. These results suggest that the mating-induced increase in AVPV/PeN Kiss1 mRNA expression may be, at least partly, involved in stimulating LH and testosterone release, and might consequently ensure male mating behavior. This study would be the first report suggesting that the AVPV/PeN kisspeptin neurons in males may play a physiological role in ensuring male reproductive performance.

Region-specific changes in brain kisspeptin receptor expression during estrogen depletion and the estrous cycle.

Kisspeptin acts as a potent neuropeptide regulator of reproduction through modulation of the hypothalamic-pituitary-gonadal axis. Previous studies revealed sex differences in brain expression patterns as well as regulation of expression by estrogen. Alternatively, sex differences and estrogen regulation of the kisspeptin receptor (encoded by Kiss1r) have not been examined at cellular resolution. In the current study, we examined whether Kiss1r mRNA expression also exhibits estrogen sensitivity and sex-dependent differences using in situ hybridization. We compared Kiss1r mRNA expression between ovariectomized (OVX) rats and estradiol (E2)-replenished OVX rats to examine estrogen sensitivity, and compared expression between gonadally intact male rats and female rats in diestrus or proestrus to examine sex differences. In OVX rats, E2 replenishment significantly reduced Kiss1r expression specifically in the hypothalamic arcuate nucleus (ARC). A difference in Kiss1r expression was also observed between diestrus and proestrus rats in the hypothalamic paraventricular nucleus (PVN), but not in the ARC. Thus, estrogen appears to have region- and context-specific effects on Kiss1r expression. However, immunostaining revealed minimal colocalization of estrogen receptor alpha (ERα) in Kiss1r-expressing neuronal populations of ARC and PVN, indicating indirect or ERα-independent regulation of Kiss1r expression. Surprisingly, unlike the kisspeptin ligand, no sexual dimorphisms were observed in either the brain distribution of Kiss1r expression or in the number of Kiss1r-expressing neurons within enriched brain nuclei. The current study reveals marked differences in regulation between kisspeptin and kisspeptin receptor, and provides an essential foundation for further study of kisspeptin signaling and function in reproduction.

Neurochemical Characterization of Neurons Expressing Estrogen Receptor ß in the Hypothalamic Nuclei of Rats Using in Situ Hybridization and Immunofluorescence.

Estrogens play an essential role in multiple physiological functions in the brain, including reproductive neuroendocrine, learning and memory, and anxiety-related behaviors. To determine these estrogen functions, many studies have tried to characterize neurons expressing estrogen receptors known as ERα and ERß. However, the characteristics of ERß-expressing neurons in the rat brain still remain poorly understood compared to that of ERα-expressing neurons. The main aim of this study is to determine the neurochemical characteristics of ERß-expressing neurons in the rat hypothalamus using RNAscope in situ hybridization (ISH) combined with immunofluorescence. Strong Esr2 signals were observed especially in the anteroventral periventricular nucleus (AVPV), bed nucleus of stria terminalis, hypothalamic paraventricular nucleus (PVN), supraoptic nucleus, and medial amygdala, as previously reported. RNAscope ISH with immunofluorescence revealed that more than half of kisspeptin neurons in female AVPV expressed Esr2, whereas few kisspeptin neurons were found to co-express Esr2 in the arcuate nucleus. In the PVN, we observed a high ratio of Esr2 co-expression in arginine-vasopressin neurons and a low ratio in oxytocin and corticotropin-releasing factor neurons. The detailed neurochemical characteristics of ERß-expressing neurons identified in the current study can be very essential to understand the estrogen signaling via ERß.

Applicability of Anti-Human Estrogen Receptor ß Antibody PPZ0506 for the Immunodetection of Rodent Estrogen Receptor ß Proteins.

Several lines of controversial evidence concerning estrogen receptor ß (ERß) remain to be solved because of the unavailability of specific antibodies against ERß. The recent validation analysis identified a monoclonal antibody (PPZ0506) with sufficient specificity against human ERß. However, the specificity and cross-reactivity of PPZ0506 antibody against ERß proteins from laboratory animals have not been confirmed. In the present study, we aimed to validate the applicability of PPZ0506 to rodent studies. The antibody exhibited specific cross-reactivity against mouse and rat ERß proteins in immunoblot and immunocytochemical experiments using transfected cells. In immunohistochemistry for rat tissue sections, PPZ0506 showed immunoreactive signals in the ovary, prostate, and brain. These immunohistochemical profiles of rat ERß proteins in rat tissues accord well with its mRNA expression patterns. Although the antibody was reported to show the moderate signals in human testis, no immunoreactive signals were observed in rat testis. Subsequent RT-PCR analysis revealed that this species difference in ERß expression resulted from different expression profiles related to the alternative promoter usage between humans and rats. In conclusion, we confirmed applicability of PPZ0506 for rodent ERß studies, and our results provide a fundamental basis for further examination of ERß functions.

Role of cAMP in phenotypic changes of osteoblasts.

Bone remodeling is precisely controlled by bone formation and bone resorption, and osteoblasts are responsible for both processes. Osteoblasts exhibit an osteoclastogenic phenotype in response to elevated intracellular cyclic AMP [cAMP]i levels. However, the role of cAMP in osteoblasts acquiring an osteogenic phenotype is controversial. To elucidate the effect of cAMP on both phenotypes, an osteoblast-like cell line, TMS-12, was established in our laboratory and used in this study. Dibutyryl-cAMP (dBcAMP), a cAMP analogue, inhibited mineralization in TMS-12 cells and MC3T3E1 cells (an osteoblast-like cell line) but promoted osteoclast-supporting activity in TMS-12 cells. Moreover, mineralization was inhibited in glucagon receptor-transduced TMS-12 cells (TMS-12GCGR) after glucagon treatment to increase endogenous [cAMP]i levels. However, the osteoclast-supporting activity of TMS-12GCGR cells was stimulated by glucagon treatment. These cAMP-induced phenotypic changes of osteoblasts were also supported by their gene expression profile. These results suggest that [cAMP]i is an important factor mediating phenotypic changes of osteoblasts. Our findings may provide valuable insights into the mechanisms that underlie bone remodeling in both, healthy and diseased states.

Suitable reference gene selection for gene expression studies in knee osteoarthritis synovium using quantitative PCR analysis.

Osteoarthritis (OA) is the leading cause of musculoskeletal disability in the elderly. Insights into the biological features of OA are obtained by characterization of the molecular features by gene expression profiling using reverse transcription-quantitative PCR (RT-qPCR). However, it has recently become evident that the use of suitable reference genes is required for appropriate normalization of this technique. Here total RNA was isolated from the synovium of 18 men and 20 women who underwent total knee arthroplasty for knee OA (KOA). We validated the expression stability of 7 candidate housekeeping genes (ACTB, B2M, GAPDH, HPRT1, RPL13A, SDHA, and YWHAZ) in the synovium of KOA with 3 commonly used algorithms (geNorm, NormFinder, and BestKeeper). Additionally, we evaluated expression profiles of the steroid hormone receptor (AR, ESR1, ESR2, GR, MR, and PR) and proinflammatory cytokines (IL1B and IL6) genes in the synovium and their correlations with the risk factors of KOA, using the most and least stable housekeeping genes for comparison. Results showed that HPRT1 was the most stable gene, whereas B2M was the least stable. RT-qPCR analysis revealed sexually dimorphic expression of AR, IL1B, and IL6; intercorrelations between steroid hormone receptor expression levels and female-specific correlations of IL1B expression with ESR1 and PR expression, IL6 expression with ESR1 and GR expression, and body mass index with AR and PR expression; and the choice of the least stable reference gene altered several correlations and statistical significances. In conclusion, HPRT1 was identified as the suitable reference gene for normalization in the OA synovium.

Distinct dynorphin expression patterns with low- and high-dose estrogen treatment in the arcuate nucleus of female rats.

Kisspeptin (KISS1; encoded by Kiss1) neurons in the arcuate nucleus (ARC) coexpress tachykinin 3 (TAC3; also known as neurokinin B) and dynorphin A (PDYN). Accordingly, they are termed KNDy neurons and considered to be crucial in generating pulsatile release of gonadotropin-releasing hormone. Accumulating evidence suggests that Kiss1 and Tac3 are negatively regulated by estrogen. However, it has not been fully determined whether and how estrogen modulates Pdyn and PDYN. Here, we examined the expression of Pdyn mRNA and PDYN by in situ hybridization and immunohistochemistry, respectively, in the ARC of female rats after ovariectomy (OVX) and OVX plus low- or high-dose beta-estradiol (E2) replacement. We also investigated the effect of E2 on expression of Kiss1, KISS1, Tac3, and TAC3. Furthermore, colocalization of PDYN and estrogen receptor alpha (ESR1) was determined. Subsequently, we found that low-dose E2 treatment had no effect on Pdyn mRNA-expressing cells, but increased PDYN-immunoreactive (ir) cell numbers. In contrast, high-dose E2 treatment resulted in prominent reductions in both Pdyn mRNA-expressing and PDYN-ir cell numbers. Changes induced by low or high doses of E2 were similarly observed in the expression of Kiss1, KISS1, Tac3, and TAC3. The majority of PDYN-ir neurons coexpressed ESR1 in all groups. Our results indicate that E2 regulates the expression of PDYN, as well as KISS1 and TAC3, with regulation by E2 differing according to its levels.

Development of an imaging system for in vivo real-time monitoring of neuronal activity in deep brain of free-moving rats.

We have newly developed a system that allows monitoring of the intensity of fluorescent signals from deep brains of rats transgenically modified to express enhanced green fluorescent protein (eGFP) via an optical fiber. One terminal of the optical fiber was connected to a blue semiconductor laser oscillator/green fluorescence detector. The other terminal was inserted into the vicinity of the eGFP-expressing neurons. Since the optical fiber was vulnerable to twisting stresses caused by animal movement, we also developed a cage in which the floor automatically turns, in response to the turning of the rat's head. This relieved the twisting stress on the optical fiber. The system then enabled real-time monitoring of fluorescence in awake and unrestrained rats over many hours. Using this system, we could continuously monitor eGFP-expression in arginine vasopressin-eGFP transgenic rats. Moreover, we observed an increase of eGFP-expression in the paraventricular nucleus under salt-loading conditions. We then performed in vivo imaging of eGFP-expressing GnRH neurons in the hypothalamus, via a bundle consisting of 3000 thin optical fibers. With the combination of the optical fiber bundle connection to the fluorescence microscope, and the special cage system, we were able to capture and retain images of eGFP-expressing neurons from free-moving rats. We believe that our newly developed method for monitoring and imaging eGFP-expression in deep brain neurons will be useful for analysis of neuronal functions in awake and unrestrained animals for long durations.

Characterization of rodent constitutively active estrogen receptor α variants and their constitutive transactivation mechanisms.

Estrogen receptor α (ERα) mRNAs exhibit remarkable heterogeneity owing to complicated alternative splicing. Some encode C-terminally-truncated ERα proteins, which display ligand-independent transactivation or dominant-negative activity. We previously characterized C-terminally-truncated ERα mRNA variants with cryptic sequences in humans and mice, and demonstrated that helices in the ligand-binding domains (LBDs) of ERα variants contribute to ligand-independent transcriptional activity. However, existence of non-conventional coding exons and generation of constitutively active ERα variants have remained to be examined in rats. To comparatively analyze modular organization and splicing profiles of the ERα genes, the range of C-terminally-truncated ERα variants was explored in rats and mice using rapid amplification of cDNA ends and RT-PCR cloning. Furthermore, their functions were determined in transiently transfected cells using expression constructs and luciferase reporter assays. Multiple cryptic exons and C-terminally-truncated ERα variant mRNAs were identified in rats and mice. Naturally occurring and artificially truncated variants/constructs lacking helix 5 potentially exhibited gain-of-function in transfected cells. Although cryptic exons and splicing profiles were poorly conserved among humans, mice, and rats, constitutively active variants were generated from the ERα genes. Moreover, the primary mechanism of ligand-independent activation by C-terminally-truncated ERα variants is C-terminus to helix 5 deletion in the LBD. This comparative study documented the complexity of ERα genes, mRNAs, and proteins, and further determined the underlying structural basis of ligand-independent activation by C-terminally-truncated ERα variants.

The increase in the number of spines on the gonadotropin-releasing hormone neuron across pubertal development in rats.

The onset of puberty is initiated by an increase in the release of the gonadotropin-releasing hormone (GnRH) from GnRH neurons in the hypothalamus. However, the precise mechanism that leads to the activation of GnRH neurons at puberty remains controversial. Spines are small protrusions on the surface of dendrites that normally receive excitatory inputs. In this study, we analyzed the number and morphology of spines on GnRH neurons to investigate changes in synaptic inputs across puberty in rats. For morphological estimation, we measured the diameter of the head (DH) of each spine and classified them into small-type (DH < 0.65 µm), large-type (DH > 0.65 µm) and giant-type (DH > 0.9 µm). The greatest number of spines was observed at the proximal dendrite within 50 µm of the soma. At the soma and proximal dendrite, the number of spines was greater in adults than in juveniles in both male and female individuals. Classification of spines revealed that the increase in spine number was due to increases in large- and giant-type spines. To further explore the relationship between spines on GnRH neurons and pubertal development, we next analyzed adult rats neonatally exposed to estradiol benzoate, in which puberty onset and reproductive functions are disrupted. We found a decrease in the number of all types of spines. These results suggest that GnRH neurons become to receive more and greater excitatory inputs on the soma and proximal dendrites as a result of the changes that occur at puberty and that alteration to spines plays a pivotal role in normal pubertal development.

Corrigendum: Macrophage SREBP1 regulates skeletal muscle regeneration.

[This corrects the article DOI: 10.3389/fimmu.2023.1251784.].

Estrogen Receptor α Isoforms Generated by Alternative Use of Cryptic Exons.

Estrogen receptor α (ERα) regulates several physiological functions. In pathophysiological conditions, ERα is involved in the development and progression of estrogen-sensitive tumors. The ERα gene contains multiple 5'-untranslated exons and eight conventional coding exons and presents multiple isoforms generated by alternative promoter usage and alternative splicing. This gene also possesses non-conventional exons in the 3'- and intronic regions, and alternative use of cryptic exons contributes to further diversity of ERα mRNAs and proteins. Recently, the genomic organization of ERα genes and the splicing profiles of their transcripts were comparatively analyzed in humans, mice, and rats, and multiple ERα isoforms with distinct structures and functions were identified. These transcripts contain cryptic sequences that encode insertion-containing or truncated ERα proteins. In particular, alternative cryptic exons with in-frame stop codons yield transcripts encoding C-terminally-truncated ERα proteins. The C-terminally-truncated ERα isoforms lack part or all of the ligand-binding domain but possess an isoform-specific sequence. Some of these isoforms exhibit constitutive transactivation and resistance to estrogen receptor antagonists. Although numerous studies have reported conflicting results regarding their functions, the critical determinant for their gain-of-function has been identified structurally. Here we review recent progress in ERα variant research concerning the genomic organization of ERα genes, splicing profiles of ERα transcripts, and transactivation properties of ERα isoforms.

Recent Advances in High-sensitivity In Situ Hybridization and Costs and Benefits to Consider When Employing These Methods.

In situ hybridization (ISH), which visualizes nucleic acids in tissues and cells, is a powerful tool in histology and pathology. Over 50 years since its invention, multiple attempts have been made to increase the sensitivity and simplicity of these methods. Therefore, several highly sensitive in situ hybridization methods have been developed that offer researchers a wide range of options. When selecting these in situ hybridization variants, their signal-amplification principles and characteristics must be understood. In addition, from a practical point of view, a method with good monetary and time-cost performance must be chosen. This review introduces recent high-sensitivity in situ hybridization variants and presents their principles, characteristics, and costs.

Central injection of neuropeptide B induces luteinizing hormone release in male and female rats.

Neuropeptide B (NPB) has been identified as an endogenous peptide ligand for the orphan receptor NPBWR1. However, the effect of NPB on the central regulatory mechanisms of reproductive functions remains unclear. Our findings indicated the presence of Npb, Npw (which is another ligand for NPBWR1), and Npbwr1 mRNA in the hypothalamus of male and female rats at each stage of the estrous cycle. Npb mRNA expression was found to be significantly higher in diestrus compared to estrus. The expression of Npw mRNA was one order of magnitude lower than that of Npb mRNA, and Npw mRNA expression in diestrus was significantly higher than that in the other stages of the estrous cycle. Furthermore, Npbwr1 mRNA expression was found to be significantly higher in diestrus compared to the other stages of the estrous cycle and intact males. Notably, estrogen did not alter the expression of Npb, Npw, and Npbwr1 mRNAs in the hypothalamus of females. Central injection of NPB increased plasma luteinizing hormone (LH) levels in both intact males and estrogen-primed ovariectomized females but not in ovariectomized females. These results suggest that NPB-NPBWR1 signaling would be a facilitatory regulatory mechanism in the reproductive function of male and female rats. To the best of our knowledge, this study is the first report to describe the central role of NPB-NPBWR1 signaling in LH regulation in mammals.