Endocrine Cell Populations Research Articles

8686 scRNA-seq of pituitary from letrozole-induced PCOS mouse model reveals abnormal prolactin secretion

Abstract Disclosure: G. De Robles: None. N. Ujagar: None. Z. Del Mundo: None. D. Nicholas: None. Polycystic ovary syndrome (PCOS) has a complex pathophysiology and is the most common endocrine disorder among reproductive-aged women. Ovarian hormonal dynamics are regulated via the secretion of the gonadotropin-releasing hormone (GnRH), which stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gland. High serum levels of LH in PCOS patients indicate an increase in GnRH levels, but the mechanisms within the anterior pituitary at a cellular level are unclear. We performed single-cell mRNA sequencing (scRNA-seq) on the pituitaries of the letrozole-induced (LET) mouse model of PCOS to profile the functional genomics of endocrine cell populations relative to serum hormone levels. The population percentage of lactotropes significantly decreased; in contrast, two gonadotrope populations significantly increased in the LET mice compared to the placebo-treated mice. Consistent with these changes, an increasing trend of serum LH levels and a decreasing trend of serum prolactin (PRL) levels were observed in the LET mice, while no change occurred in serum growth hormone levels. A gene set enrichment analysis (GSEA) on the differentially expressed genes (DEGs) from the lactotropes revealed an association with negative regulation of protein kinase activity. These DEGs included Hspb1, Park7, Pcp4, and Ppia, which all share the function of mediating stress-induced apoptosis. A GSEA on the DEGs between the two gonadotrope clusters in the LET mice also confirmed a variation in biological pathways linked to ribosomal subunits, transcription regulation, and the regulation of steroid metabolic processes. Based on the scRNA-seq results of the anterior pituitaries of LET mice, alterations in the proportions of hormone-secreting cells may be a response to hyperandrogenism, in which functionally diverse gonadotropes could contribute to heterogeneity in reproductive phenotypes. Changes in the lactotrope population and PRL serum levels also support further research on the lactation hormone and its role in PCOS. PRL is involved in metabolic homeostasis and reproduction, and understanding its dysregulations may provide additional insight into the pathophysiology of PCOS. Presentation: 6/3/2024

Hyperglycemia influences the cell proliferation and death of the rat endocrine pancreas in the neonatal period

AimsTo evaluate the cell proliferation and death, and structural morphology of the pancreatic islet cells of the rats with hyperglycemia in the first month of life and compare to those of the control rats. Main methodsFemale Sprague-Dawley newborn rats received Streptozotocin (a beta-cytotoxic drug) at birth for diabetes induction. Control and hyperglycemic animals were euthanized on different days of life: 5, 10, 15, and 30. The pancreas was collected and processed for immunohistochemical analysis of cleaved Caspase-3 (cell death), Ki-67 (cell proliferation), PDX-1 (transcription factor responsible for insulin synthesis), and endocrine hormones (insulin, glucagon, and somatostatin). Key findingsControl females showed a higher percentage (%) of Ki-67-positive(+) cells on D10 and D15, a higher % of insulin+ and somatostatin+ cells on D15 and D30, a lower % of PDX-1+ cells on D10, and a higher % of glucagon+ cells on D10 and D30. Hyperglycemic females showed a lower % of Ki-67+ cells on D15, a higher % of cleaved Caspase-3+ cells on D15, and insulin+ cells on D15 and D30. In the comparison among the experimental groups, the hyperglycemic females showed an increased % of cleaved Caspase-3+ and Ki-67+ cells and a lower % of PDX-1+ cells. SignificanceThis study enabled a better understanding of the abnormal pancreas development regarding cellular proliferation, apoptosis, and hormonal synthesis in the neonatal period. Thus, the pancreatic islets of hyperglycemic rats do not reestablish the normal endocrine cell population, and cellular apoptosis overcame the proliferative activity of these cells.

Extra-islet cells expressing insulin or glucagon in the pancreas of young organ donors

AimsThe existence of insulin- or glucagon-expressing extra-islet endocrine cells scattered in the pancreas is well-known, but they have been sparsely characterized. The aim of this study was to examine their density, distribution, transcription-factor expression, and mitotic activity in young non-diabetic subjects.MethodsMultispectral imaging was used to examine PDX1, ARX, Ki67, insulin and glucagon in extra-islet endocrine cells in pancreatic tissue from organ donors aged 1–25 years.ResultsExtra-islet insulin- or glucagon-positive cells were frequent in all donors (median 17.3 and 22.9 cells/mm2 respectively), with an insulin:glucagon cell ratio of 0.9. The density was similar regardless of age. PDX1 localized mainly to insulin-, and ARX mainly to glucagon-positive cells but, interestingly, many of the cells were negative for both transcription factors. Double-hormone-positive cells were rare but found in all age groups, as were insulin-positive cells expressing ARX and glucagon-positive cells expressing PDX1. Extra-islet endocrine cells with Ki67 expression were present but rare (0–2%) in all age groups.ConclusionsExtra-islet endocrine cells are more frequent than islets. The preserved extra-islet cell density during pancreas volume-expansion from childhood- to adulthood indicates that new cells are formed, possibly from replication as cells with mitotic activity were discovered. The lack of transcription-factor expression in many cells indicates that they are immature, newly formed or plastic. This, together with the mitotic activity, suggests that these cells could play an important role in the expansion of beta-cell mass in situations of increasing demand, or in the turnover of the endocrine cell population.

Forward programming of hiPSCs towards beta-like cells using Ngn3, Pdx1, and MafA

Transplantation of stem cell-derived β-cells is a promising therapeutic advancement in the treatment of type 1 diabetes mellitus. A current limitation of this approach is the long differentiation timeline that generates a heterogeneous population of pancreatic endocrine cells. To address this limitation, an inducible lentiviral overexpression system of mature β-cell markers was introduced into human induced-pluripotent stem cells (hiPSCs). Following the selection of the successfully transduced hiPSCs, the cells were treated with doxycycline in the pancreatic progenitor induction medium to support their transition toward the pancreatic lineage. Cells cultured with doxycycline presented the markers of interest, NGN3, PDX1, and MAFA, after five days of culture, and glucose-stimulated insulin secretion assays demonstrated that the cells were glucose-responsive in a monolayer culture. When cultured as a spheroid, the markers of interest and insulin secretion in a static glucose-stimulated insulin secretion assay were maintained; however, insulin secretion upon consecutive glucose challenges was limited. Comparison to human fetal and adult donor tissues identified that although the hiPSC-derived spheroids present similar markers to adult insulin-producing cells, they are functionally representative of fetal development. Together, these results suggest that with optimization of the temporal expression of these markers, forward programming of hiPSCs towards insulin-producing cells could be a possible alternative for islet transplantation.

Abstract 434: Understanding metabolic heterogeneity of estrogen receptor positive metastatic breast tumors using spatial transcriptomics and geographic information systems approaches

Abstract The tumor microenvironment consists of numerous cell populations showing diverse metabolic preferences, causing metabolic heterogeneity and therapy resistance. Fulvestrant, a clinically approved selective estrogen receptor (ER) degrader, is the standard of care for ER-positive breast cancer, yet resistance has been reported among metastatic breast cancer patients. In our previous studies, we showed such resistance is attributable to metabolic heterogeneity in the tumor microenvironment. Despite metabolic heterogeneity posing a common challenge in cancer research, current methods, such as histology and Magnetic Resonance Imaging, cannot explore nutrient uptake at transcript-level resolution with spatial context, making it difficult to understand the heterogeneity that affects cellular growth and metabolism. Visium Spatial Transcriptomics, a method that shows regional gene expression, is emerging as a powerful spatial analysis tool, and we integrated this method with geographic information systems to investigate the tumor microenvironment heterogeneity and highlight the importance of cell-microenvironment interactions. We developed a computational pipeline to visualize hotspots of spatially relevant genes using breast cancer metastatic samples from xenograft models and human patients. This pipeline integrates Visium tools and geospatial methods for processing transcriptome profiling data. We analyzed the degree of local spatial association based on Local Moran’s I to estimate regions with significant co-localization of single and multiple gene expressions within a tumor. We then leveraged the co-localized areas in the process of biomarker identification and revealing biological pathways. We also used unsupervised clustering of the complete transcriptome profiles to test the resemblance between identified clusters and hotspots as well as any disruption in the spatial structure of clusters in response to therapy or metastasis. Gene set enrichment analysis of these clusters revealed several glucose or lipid metabolism pathways, and mapping of the genes corresponding to each pathway showed mutually exclusive gene expression patterns in the sample. We also observed minimal co-localization of two metabolic pathways but extensive co-localization between metabolic genes and ER target genes, suggesting distinct metabolic preferences and spatial distributions of endocrine therapy resistant cell populations. Altogether, our analysis pipeline established a computational framework for characterizing the link between tumor microenvironment, local metabolism, and changes in transcriptional response to external stimuli. Our findings support the development of strategies to target different endocrine resistant regions using a combination of ER and metabolic signaling inhibitors. Citation Format: Jin Young Yoo, Sabrina Akter, Nandana Varma, Atharva Jain, Aiman Soliman, Zeynep Madak Erdogan. Understanding metabolic heterogeneity of estrogen receptor positive metastatic breast tumors using spatial transcriptomics and geographic information systems approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 434.

Effect of quercetin administration on electron microscopic changes in testicular interstitial endocrinocytes during long-term central blockade of luteinising hormone in rats

Quercetin is a flavonoid with potential health benefits and it may help prevent cardiovascular diseases, reduce the risk of degenerative brain processes and cancer, and has antioxidant properties that neutralise free radicals. Substances in this group also have antioxidant properties, which help the body protect itself from the harmful effects of free radicals by neutralising these unstable molecules. Research indicates that the impact of free radicals on cell structure is associated with the development of chronic diseases such as diabetes, cancer, and cardiovascular pathologies. The aim of the study was to investigate the effect of quercetin administration on electron microscopic changes in the interstitial endocrinocytes of the testes under long-term central blockade of the synthesis of luteinising hormone by tryptorelin. The experiment involved 35 sexually mature male white rats. They were divided into two groups: the control group (I) received saline, while group II received subcutaneous injections of tryptorelin at a dose of 0.3 mg of active ingredient per kg of rat body weight to induce experimental central deprivation of luteinising hormone synthesis and additionally, quercetin was administered three times a day by gastric tube in terms of body weight. The study has demonstrated that the administration of tryptorelin results in structural and functional changes in the connective tissue components of rat testes. Specifically, there are quantitative and qualitative disorders in the population of interstitial endocrine cells, as well as electron microscopic changes at the subcellular level. Various pathological changes and abnormalities in the functional activity of the internal components of the cell were detected on days 270 and 365 of the experiment. The frequency, number, and size of Reinke crystals in relation to the cell volume correlated with changes in cells and increased at later stages of the study. A similar correlation with testosterone levels has not been found in the literature, which leads us to classify Reinke crystals as the result of degenerative processes in the cell. Thus, additional administration of quercetin reduces the adverse effect of tryptorelin and delays the onset of changes in the structure of interstitial endocrinocytes from day 180 to later observation periods.

NEUROD1 reinforces endocrine cell fate acquisition in pancreatic development

NEUROD1 is a transcription factor that helps maintain a mature phenotype of pancreatic β cells. Disruption of Neurod1 during pancreatic development causes severe neonatal diabetes; however, the exact role of NEUROD1 in the differentiation programs of endocrine cells is unknown. Here, we report a crucial role of the NEUROD1 regulatory network in endocrine lineage commitment and differentiation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate that Neurod1 inactivation triggers a downregulation of endocrine differentiation transcription factors and upregulation of non-endocrine genes within the Neurod1-deficient endocrine cell population, disturbing endocrine identity acquisition. Neurod1 deficiency altered the H3K27me3 histone modification pattern in promoter regions of differentially expressed genes, which resulted in gene regulatory network changes in the differentiation pathway of endocrine cells, compromising endocrine cell potential, differentiation, and functional properties.

1739-P: Ontogeny of Bihormonal Islet Cells within the Human Pancreas

Bi-hormonal islet endocrine cells have been proposed to represent an intermediate state during cellular trans-differentiation, enabling plasticity of islet beta-cells in response to metabolic stress. Beta-cell plasticity and regenerative capacity are thought to decrease with age. We investigated bi-hormonal islet endocrine cell populations present throughout the human lifespan between 11 days to 79 years of age. Immunohistochemical staining was performed on paraffin-embedded sections of pancreata from 24 individuals without diabetes for insulin, glucagon, and somatostatin. The sections were imaged with epifluorescence microscopy, and hormone-positive cells were manually counted using ImageJ software considering cells where more than 50% of the area was immunopositive. The mean proportional presence of glucagon-, insulin-, and somatostatin-immunopositive cells within islets was 28.8%, 65.1%, and 13.8% respectively, similar to previous reports for human islets. The relative presence of alpha- and beta-cells increased, and for delta-cells decreased, with advancing age. With respect to bi-hormonal cells the overall presence of glucagon+/insulin+ cells was 3.3%, for somatostatin+/insulin+ cells was 2.4%, and for glucagon+/somatostatin+ cells was 1.7% relative to total islet cell number. The proportion of glucagon+/somatostatin+ cells in the islets of female individuals, in particular, diminished with age (r2=0.43), whereas the other cell types remained largely constant with age in both sexes. The prevalence of tri-hormonal islet cells was low (0.45%). The findings suggest that bi-hormonal islet cells are present within human pancreatic islets throughout life, perhaps reflecting an ongoing potential for endocrine cell plasticity. Disclosure J.Hahm: None. J.A.Fernandez andrade: None. E.Arany: None. D.J.Hill: None. Funding Lawson Foundation

Single-cell transcriptomic atlas of parathyroid adenoma and parathyroid carcinoma.

Primary hyperparathyroidism is typically characterized by monoclonal parathyroid tumors that secrete an excessive amount of parathyroid hormone (PTH). However, the underlying pathogenesis of tumorigenesis remain unclear. We performed single-cell transcriptomic analysis on five parathyroid adenomas (PA) and two PC samples. A total of 63909 cells were divided into 11 different cell categories; endocrine cells accounted for the largest proportion of cells in both PA and PC, and patients with PC had larger populations of endocrine cells. Our results revealed significant heterogeneity in PA and PC. We identified cell cycle regulators may play a critical role in the tumorigenesis of PC. Furthermore, we found that the tumor microenvironment in PC was immunosuppressive, and endothelial cells had the highest interactions with other cell types, such as fibroblast-musculature cells and endocrine cells. PC development may be stimulated by fibroblast-endothelial cell interactions. Our study clarifies the transcriptional signatures that underlie parathyroid tumors and offer a potential significant contribution in the study of pathogenesis of PC. This article is protected by copyright. All rights reserved.

Curcumin effects on Leydig cell functions and potential therapeutic uses.

Curcumin has been ascribed with countless therapeutic effects, but its impact on testicular function has been scarcely researched. Leydig cells comprise the androgen-secreting population of the testis and may give rise to Leydig cell tumours (LCTs). Due to their steroid-secreting nature, LCTs entail endocrine, reproductive, and psychological disorders. Approximately 10% are malignant and do not respond to chemotherapy and radiotherapy. The aim of this study was to assess curcumin's impact on Leydig cells' functions and its potential effect on LCT growth. In vitro assays on MA-10 Leydig cells showed that curcumin (20-80 µmol/L) stimulates acute steroidogenesis, both in the presence and absence of db-cAMP. This effect is accompanied by an increase in StAR expression. Regarding curcumin's in vitro cytostatic capacity, we show that 40-80 µmol/L curcumin reduces MA-10 Leydig cells' proliferative capacity, which could be explained by the arrest in G2/M and the reduced viability due to the activation of the apoptotic pathway. Finally, CB6F1 mice were inoculated with MA-10 cells to generate ectopic LCT in both flanks. They received i.p. injections of 20 mg/kg curcumin or vehicle every other day for 15 days. We unveiled curcumin's capacity to inhibit LCT growth as evidenced by reduced tumour volume, weight, and area under the growth curves. No detrimental effects on general health parameters or testicular integrity were observed. These results provide novel evidence of curcumin's effects on the endocrine cell population of the testis and propose this natural compound as a therapeutic agent for LCT.

Characterization of stem-cell-derived islets during differentiation and after implantation

Recapitulation of embryonic pancreatic development has enabled development of methods for invitro islet cell differentiation using human pluripotent stem cells (hPSCs), which have the potential to cure diabetes. Advanced methods for optimal generation of stem-cell-derived islets (SC-islets) has enabled successful diabetes reversal in rodents and shown promising early clinical trial outcomes. The main impediment for use of SC-islets is concern about safety because of off-target growth resulting from contaminated residual cells. In this review, we summarize the different endocrine and non-endocrine cell populations that have been described to emerge throughout β cell differentiation and after transplantation. We discuss the most recent approaches to enrich endocrine populations and remove off-target cells. Finally, we discuss the critical quality control and release criteria testing that we anticipate will be required prior to transplantation to ensure product safety.

A New Perspective on Regulation of Pituitary Plasticity: The Network of SOX2-Positive Cells May Coordinate Responses to Challenge.

Plasticity of function is required for each of the anterior pituitary endocrine axes to support alterations in the demand for hormone with physiological status and in response to environmental challenge. This plasticity is mediated at the pituitary level by a change in functional cell mass resulting from a combination of alteration in the proportion of responding cells, the amount of hormone secreted from each cell, and the total number of cells within an endocrine cell population. The functional cell mass also depends on its organization into structural and functional networks. The mechanisms underlying alteration in gland output depend on the strength of the stimulus and are axis dependent but in all cases rely on sensing of output of the functional cell mass and its regulation. Here, we present evidence that the size of pituitary cell populations is constrained and suggest this is mediated by a form of quorum sensing. We propose that pituitary cell quorum sensing is mediated by interactions between the networks of endocrine cells and hormone-negative SOX2-positive (SOX2+ve) cells and speculate that the latter act as both a sentinel and actuator of cell number. Evidence for a role of the network of SOX2+ve cells in directly regulating secretion from multiple endocrine cell networks suggests that it also regulates other aspects of the endocrine cell functional mass. A decision-making role of SOX2+ve cells would allow precise coordination of pituitary axes, essential for their appropriate response to physiological status and challenge, as well as prioritization of axis modification.

1459-P: Human Islet Cell Transcriptome Analysis Using Single Nucleus RNA-seq Reveals New Beta-Cell Markers and Define Distinct Beta-Cell Subpopulations with Different Transcriptional Activity

Single-cell RNA sequencing (scRNA-seq) of human islets requires cell dissociation and does not provide information on the transcriptional status of islet cells. On the other hand, single-nucleus RNA sequencing (snRNA-seq) does not require cell dissociation and provides abundant information on intronic sequences that can be used to identify actively transcribed genes. Based on this, we seek to compare scRNA-seq and snRNA-seq approaches in human islets to determine: 1) whether similar cell clusters could be detected; 2) whether new gene markers could be discovered in human islet endocrine cells using intronic reads; 3) whether human beta cell subpopulations could be identified based on INS expression dynamics; and 4) whether snRNA-seq could be used in human islet grafts transplanted in immunosuppressed mice. We performed scRNA-seq and snRNA-seq on three pairs of human islets obtained from three healthy adult human donors using exon only or exon plus intron reads, respectively. Analysis of integrated data revealed similar human islet cell clusters. In the snRNA-seq data, however, top differentially expressed genes were identified as new markers of human endocrine cells such as ZNF385D (β-cells) , PTPRT (α-cells) , LRFN5 (δ-cells) and CACNA2D3 (PP cells) . These markers also accurately define endocrine cell populations in human islet grafts. Additionally, we distinguished several beta cell sub-clusters- INS rich cluster, HNRNPA2B1 (vesicle) rich cluster and active INS transcribing cluster. In conclusion, snRNA-seq analysis of human islet cells is a previously unrecognized tool for the identification of human islet cell types in samples where nuclear RNA processing is required. By comparing INS expression between scRNA-seq and snRNA-seq data sets, we can detect different beta cell sub-populations with distinct gene expression patterns representing different biological dynamic states. Disclosure R.Kang: None. Y.Li: None. C.Rosselot: None. D.Scott: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. G.Lu: None. Funding NIH DK105015

Anatomical and histological characteristics of pituitary gland in domestic animals

As it regulates important physical functions and general wellbeing, the hypophysis plays a major role in responding to the increased demand for different hormones. In the current review, we described the anatomical and the histological structures of the pituitary glands in different domestic animals. Unlike other glands, the pituitary gland has two main parts; the adenohypophysis (anterior lobe), which contains multiple populations of endocrine cells and is responsible for producing each of the pituitary trophic hormones. Another part of the pituitary gland is that the neurohypophysis (posterior lobe) secretes granules that contain certain neurohypophyseal hormones, like antidiuretic hormone and oxytocin, which are synthesized in the hypothalamus.

Whole-slide imaging and a Fiji-based image analysis workflow of immunohistochemistry staining of pancreatic islets

Quantification of cell populations in tissue sections is frequently examined in studies of human disease. However, traditional manual imaging of sections stained with immunohistochemistry is laborious, time-consuming, and often assesses fields of view rather than the whole tissue section. The analysis is usually manual or utilises expensive proprietary image analysis platforms. Whole-slide imaging allows rapid automated visualisation of entire tissue sections. This approach increases the quantum of data generated per slide, decreases user time compared to manual microscopy, and reduces selection bias. However, such large data sets mean that manual image analysis is no longer practicable, requiring an automated process. In the case of diabetes, the contribution of various pancreatic endocrine cell populations is often investigated in preclinical and clinical samples. We developed a two-part method to measure pancreatic endocrine cell mass, firstly describing imaging using an automated slide-scanner, and secondly, the analysis of the resulting large image data sets using the open-source software, Fiji, which is freely available to all researchers and has cross-platform compatibility. This protocol is highly versatile and may be applied either in full or in part to analysis of IHC images created using other imaging platforms and/or the analysis of other tissues and cell markers.

Sleeve Gastrectomy and Roux-En-Y Gastric Bypass. Two Sculptors of the Pancreatic Islet

Several surgical procedures are performed for the treatment of obesity. A main outcome of these procedures is the improvement of type 2 diabetes mellitus. Trying to explain this, gastrointestinal hormone levels and their effect on organs involved in carbohydrate metabolism, such as liver, gut, muscle or fat, have been studied intensively after bariatric surgery. These effects on endocrine-cell populations in the pancreas have been less well studied. We gathered the existing data on these pancreatic-cell populations after the two most common types of bariatric surgery, the sleeve gastrectomy (SG) and the roux-en-Y gastric bypass (RYGB), with the aim to explain the pathophysiological mechanisms underlying these surgeries and to improve their outcome.

Characterisation of Ppy-lineage cells clarifies the functional heterogeneity of pancreatic beta cells in mice

Aims/hypothesisPancreatic polypeptide (PP) cells, which secrete PP (encoded by the Ppy gene), are a minor population of pancreatic endocrine cells. Although it has been reported that the loss of beta cell identity might be associated with beta-to-PP cell-fate conversion, at present, little is known regarding the characteristics of Ppy-lineage cells.MethodsWe used Ppy-Cre driver mice and a PP-specific monoclonal antibody to investigate the association between Ppy-lineage cells and beta cells. The molecular profiles of endocrine cells were investigated by single-cell transcriptome analysis and the glucose responsiveness of beta cells was assessed by Ca2+ imaging. Diabetic conditions were experimentally induced in mice by either streptozotocin or diphtheria toxin.ResultsPpy-lineage cells were found to contribute to the four major types of endocrine cells, including beta cells. Ppy-lineage beta cells are a minor subpopulation, accounting for 12–15% of total beta cells, and are mostly (81.2%) localised at the islet periphery. Unbiased single-cell analysis with a Ppy-lineage tracer demonstrated that beta cells are composed of seven clusters, which are categorised into two groups (i.e. Ppy-lineage and non-Ppy-lineage beta cells). These subpopulations of beta cells demonstrated distinct characteristics regarding their functionality and gene expression profiles. Ppy-lineage beta cells had a reduced glucose-stimulated Ca2+ signalling response and were increased in number in experimental diabetes models.Conclusions/interpretationOur results indicate that an unexpected degree of beta cell heterogeneity is defined by Ppy gene activation, providing valuable insight into the homeostatic regulation of pancreatic islets and future therapeutic strategies against diabetes.Data availabilityThe single-cell RNA sequence (scRNA-seq) analysis datasets generated in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession number GSE166164 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE166164).Graphical abstract

3D Atlas of the Pituitary Gland of the Model Fish Medaka (Oryzias latipes).

In vertebrates, the anterior pituitary plays a crucial role in regulating several essential physiological processes via the secretion of at least seven peptide hormones by different endocrine cell types. Comparative and comprehensive knowledge of the spatial distribution of those endocrine cell types is required to better understand their physiological functions. Using medaka as a model and several combinations of multi-color fluorescence in situ hybridization, we present the first 3D atlas revealing the gland-wide distribution of seven endocrine cell populations: lactotropes, thyrotropes, Lh and Fsh gonadotropes, somatotropes, and pomca-expressing cells (corticotropes and melanotropes) in the anterior pituitary of a teleost fish. By combining in situ hybridization and immunofluorescence techniques, we deciphered the location of corticotropes and melanotropes within the pomca-expressing cell population. The 3D localization approach reveals sexual dimorphism of tshba-, pomca-, and lhb-expressing cells in the adult medaka pituitary. Finally, we show the existence of bi-hormonal cells co-expressing lhb-fshb, fshb-tshba and lhb-sl using single-cell transcriptomics analysis and in situ hybridization. This study offers a solid basis for future comparative studies of the teleost pituitary and its functional plasticity.

Single-Cell RNA Sequencing Analysis of Chicken Anterior Pituitary: A Bird's-Eye View on Vertebrate Pituitary.

It is well-established that anterior pituitary contains multiple endocrine cell populations, and each of them can secrete one/two hormone(s) to regulate vital physiological processes of vertebrates. However, the gene expression profiles of each pituitary cell population remains poorly characterized in most vertebrate groups. Here we analyzed the transcriptome of each cell population in adult chicken anterior pituitaries using single-cell RNA sequencing technology. The results showed that: (1) four out of five known endocrine cell clusters have been identified and designated as the lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs, respectively. Somatotrophs were not analyzed in the current study. Each cell cluster can express at least one known endocrine hormone, and novel marker genes (e.g., CD24 and HSPB1 in lactotrophs, NPBWR2 and NDRG1 in corticotrophs; DIO2 and SOUL in thyrotrophs, C5H11ORF96 and HPGDS in gonadotrophs) are identified. Interestingly, gonadotrophs were shown to abundantly express five peptide hormones: FSH, LH, GRP, CART and RLN3; (2) four non-endocrine/secretory cell types, including endothelial cells (expressing IGFBP7 and CFD) and folliculo-stellate cells (FS-cells, expressing S100A6 and S100A10), were identified in chicken anterior pituitaries. Among them, FS-cells can express many growth factors, peptides (e.g., WNT5A, HBEGF, Activins, VEGFC, NPY, and BMP4), and progenitor/stem cell-associated genes (e.g., Notch signaling components, CDH1), implying that the FS-cell cluster may act as a paracrine/autocrine signaling center and enrich pituitary progenitor/stem cells; (3) sexually dimorphic expression of many genes were identified in most cell clusters, including gonadotrophs and lactotrophs. Taken together, our data provides a bird’s-eye view on the diverse aspects of anterior pituitaries, including cell composition, heterogeneity, cell-to-cell communication, and gene expression profiles, which facilitates our comprehensive understanding of vertebrate pituitary biology.

Вікові особливості топографії і кількості ендокриноцитів кишечника свійської качки

The aim of the study was to determine the features of the topography and the number of the total population of endocrinocytes and enterochromaffin cells in the intestines of ducks during the first year of the postnatal period of ontogenesis. The experiments were performed on domestic ducks (Anas platyrhynchos domesticus) of black white-breasted breed (n = 5) 9 groups: 1-, 3-, 7-, 14-, 21-days-old, 1-, 2-, 6-month-old and 1-year-old. Determination of the total population of endocrine cells (argyrophilic) and enterochromaffin (argentaffin) was performed on histological specimens made from cross-sections of the middle section of the duodenum, jejunum, ileum, cecum and rectum, stained by Masson and Grimelius. The endocrine cells of 1- and 3-days-old ducks had a «superficial» location due to insufficient formation of crypts and villi. In older birds, in the duodenum, jejunum, and ileum, both argyrophilic and argentaffin APUD cells were localized mainly in crypts, and in the cecum and rectum, they were also in the epithelial layer of the villi. During the entire observation period, the highest content of both argyrophilic and argentaffin endocrinocytes in all intestines were found in 21-days-old ducks. Already at 3-days-old age, the total number of endocrine cells in various intestines corresponded to those of an adult bird. The highest number of argyrophilic and argentaffin endocrinocytes was found in the ileum and cecum, less in the rectum and jejunum, and the smallest number was in the duodenum. Enterochromaffin cells are the most common type of endocrinocytes in the ducks’ intestines, their relative content ranged from 40.1 to 97.5%. The highest relative content of argentaffin cells at 1-, 3- and 7-day-old was observed in the duodenum, at 14-day-old – in the duodenum and colon, at 21-day-old – in the rectum, at 1- and 2-month-old in jejunum, at the age of 6 month – in the duodenum, jejunum and caecum, and at the age of 1-year-old – in the ileum and cecum. In addition to argyrophilic endocrinocytes, which were located in the epithelium, other cells were found in the loose connective tissue of the villi base, the cytoplasm of which was also intensively impregnated with silver nitrate. Such cells were smaller and mostly oval in shape. Their cytoplasm was impregnated almost evenly around the nucleus and had no polarity in the location of the granules. Probably those were mast cells, which also contain bioamines, and, as well as enteroendocrinocytes, are found in the argyrophilic reaction.