Single Nucleus Transcriptome and Chromatin Accessibility Landscapes of Human Pituitaries

The pituitary gland regulates key physiological functions, including growth, sexual maturation, reproduction, and lactation. Here, we present a paired single-nuclei (sn) transcriptome and chromatin accessibility characterization of six post-mortem human pituitaries. These samples were from juvenile, adult, and elderly male and female subjects. Well-correlated snRNAseq and snATACseq datasets facilitated robust identification of the major pituitary cell types in each sample. Using latent variable pathway analysis, we uncovered previously unreported coordinated gene expression modules and chromatin accessibility programs for each major cell type as well as an age-specific program across all the endocrine cell types. These largely appear to be congruent between human and mouse datasets. Given the importance of murine models in the study of human pituitary disorders and pituitary physiology, we next sought to compare expression profiles of pituitary cell types in mouse vs. human. Murine and human cell types were well correlated, exemplified by coordinated gene expression programs, especially for undifferentiated stem cells (SCs). In both species, we identified clusters corresponding to naive and committing SCs. All human SC clusters expressed the established SC markers SOX2 and SOX9, as well as genes involved in SC regulatory pathways (WWTR1, YAP1 andPITX2). Additional markers previously reported in murine pituitary SCs were also found in human SC, including WIF1, LGR5, FOS, CDH1, EGFR, LGR4, and WLS. Remarkably, in human, the main naive SC cluster was roughly divided into a high-JUN and a low-JUN expressing subgroup, whereas Jun expression was less pronounced in the murine SC cluster. In both species, committing SC clusters expressed the endocrine markers for POU1F1, TSHB, or POMC, while SCs committing to an intermediate lobe/melanotrope cell identity were distinguishable based on PAX7 expression. In addition, in the human datasets we identify a population of cells as originating from the pars tuberalis. We offer a range of markers that can be utilized for in vivo validation of these cells. Overall, the characterization of the murine and human pituitary SCs strongly suggests the co-existence of subpopulations with different lineage commitments in addition to a single uncommitted SC population. This sn atlas of the human pituitary is a valuable resource that will be made web-accessible.

Ultrastructural Changes of Melanotropic Cells of the Intermediate Share of the Adenogophysis in the Age Aspect

In modern theoretical and practical biology and medicine, the key problem of research is to reveal the patterns of structural and functional organization of the human and animal body at different stages of development. Literature sources provide very limited data on the organization of the intermediate lobe of the adenohypophysis in humans, leaving insufficiently studied the ultrastructural state and activity of melanotropic cells of the intermediate lobe of the adenohypophysis in animals of different ages, including rats. The issues of the intermediate pituitary gland functioning remain important and little studied, especially in the period of the beginning of the optic-thalamic system. The purpose of the research was to study the ultrastructural changes of melanotropic cells of the intermediate lobe of the adenohypophysis in rats of different ages in the norm. Material and methods. In accordance with the purpose of the study we conducted the experiment on 30 nonlinear white male rats of different ages: 14-, 45- and 90-day-old. The animals were kept in the vivarium in equivalent conditions. The keeping and using of animals was carried out in accordance with the provisions of the "General Ethical Principles of Animal Experiments", approved by the IV National Congress of Bioethics. While examining the intermediate pituitary gland of intact rats on the electron microscope, the material was fixed in 2.5% solution of glutaraldehyde on phosphate buffer with fixation in 1% solution of osmium tetroxide according to Caulfield. It was dehydrated in alcohols of increasing concentration (70%, 80%, 90%, 100%) and acetone, poured into a mixture of epon-araldite. Semi-thin sections were made from the obtained blocks, which were stained with toluidine blue. Results and discussion. At the ultrastructural level in the intermediate lobe of the adenohypophysis of 14-day-old intact rats, several cell types could be identified that differed in the number and size of secretory granules. The ultrastructure of glandular cells of 45-day-old rats had no significant differences compared with 14-day-old animals. Secretory granules of different sizes and electron densities were observed in the cytoplasm of melanotropic cells. It was often possible to see a glandular cell with numerous secretory granules in one part of the cytoplasm, while in another they were virtually absent. Most melanotropocytes were characterized by slightly compacted mitochondria, but their numbers were slightly higher than in 45-day-old rats. The nuclei of most cells were large, oval in shape with a clear structure of nucleoli and their ribosomal component. The latter represented groups of ribosomes that were collected in osmophilic complexes. The amount of heterochromatin exceeded euchromatin and it was located mainly in the membrane with areas of rarefaction in the pore area Conclusion. In 14-day-old intact male rats, the intermediate lobe of the adenohypophysis was presented as a formed functionally active organ. In the ultrastructure of the cytoplasm of melanotropes there was a moderate development of organelles, and judging by the number and size of secretory granules, as well as the density of their content, we can assume that all these cells differed from each other in their functional activity. In 45-day-old intact rats, accumulation of secretory granules was observed in the cytoplasm of melanotropes, especially near the nucleus, which indicated an increase in melanocyte-stimulating hormones synthesis with age. The ultrastructural state of the cytoplasm and nucleus also indicated an increase in functional activity. The ultrastructure of the intermediate lobe cells of the adenohypophysis of rats at the age of 90 days differed from that of younger animals by signs of different functional activity of individual melanotropic cells

MECHANISMS IN ENDOCRINOLOGY: Pioneer transcription factors in pituitary development and tumorigenesis

Pioneer transcription factors have key roles in development as master regulators of cell fate specification. Only a small fraction of all transcription factors have the pioneer ability that confers access to target genomic DNA sites embedded in so-called ‘closed’ heterochromatin. This ability to seek and bind target sites within the silenced portion of the epigenome is the basis for their role in changing cell fate. Upon binding heterochromatin sites, pioneer factors trigger remodeling of chromatin from a repressed into an active organization. This action is typically exerted at enhancer regulatory sequences, thus allowing activation of new gene subsets. During pituitary development, the only pioneer with a well-documented role is Pax7 that specifies the intermediate lobe melanotrope cell fate. In this review, a particular focus is placed on this Pax7 function but its properties are also considered within the general context of pioneer factor action. Given their potent activity to reprogram gene expression, it is not surprising that many pioneers are associated with tumor development. Overexpression or chromosomal translocations leading to the production of chimeric pioneers have been implicated in different cancers. We review here the current knowledge on the mechanism of pioneer factor action.

SOX2 is required independently in both stem and differentiated cells for pituitary tumorigenesis in p27 null mice

Loss of P27 predominantly results in development of murine pituitary intermediate lobe (IL) tumours. We previously showed that the pleiotropic protein P27 can drive repression of the transcription factor Sox2. This interaction plays an important role during development of p27-/- IL tumours because loss of one copy of Sox2 diminishes tumorigenesis. Here, we have explored the cellular origin and mechanisms underlying melanotroph tumorigenesis in p27-/- IL. We show that IL hyperplasia is associated with reduced cellular differentiation, while levels of SOX2 increase in both stem cells (SC) and melanotrophs. Using loss-of-function and lineage tracing approaches, we demonstrate that SOX2 is required cell-autonomously in p27-/- melanotrophs and SCs for tumorigenesis. This is supported by studies deleting the Sox2 regulatory region 2 (Srr2), which is the target of P27 repressive action. Single cell transcriptomic analysis reveals that activation of a SOX2-dependent MAPK pathway in SCs is important for p27-/- tumorigenesis. Our data highlight different roles of SOX2 following loss of p27, according to the cellular context. Furthermore, we uncover a tumor-promoting function for SCs, which is SOX2-dependant. In conclusion, our results imply that targeting SCs, in addition to tumour cells themselves, may represent an efficient anti-tumoral strategy in certain contexts.

Sellar Colloid Cyst—An Anatomically Aberrant and Diagnostically Challenging Entity: Report of a Rare Case with Literature Review

The sellar colloid cyst is a rare entity anatomically occurring at the intermediate lobe of the pituitary gland. Clinically a cystic sellar lesion with pressure effects usually evokes the suspicion of a pituitary adenoma. We present the case of a middle-aged woman presenting with visual diminution and bilateral optic atrophy, caused by a large sellar lesion, variably intense on magnetic resonance imaging (MRI). The subsequent histologic diagnosis of a colloid cyst was unexpected. This report highlights the subtle intraoperative and diagnostic features key to diagnosing this rarity. We also discuss a practical differential diagnostic approach relevant to the practicing surgeon and review the existing literature.

Single-cell molecular and cellular architecture of the mouse neurohypophysis

The neurohypophysis (NH), located at the posterior lobe of the pituitary, is a major neuroendocrine tissue, which mediates osmotic balance, blood pressure, reproduction, and lactation by means of releasing the neurohormones oxytocin and arginine-vasopressin from the brain into the peripheral blood circulation. The major cellular components of the NH are hypothalamic axonal termini, fenestrated endothelia and pituicytes, the resident astroglia. However, despite the physiological importance of the NH, the exact molecular signature defining neurohypophyseal cell types and in particular the pituicytes, remains unclear. Using single cell RNA sequencing, we captured seven distinct cell types in the NH and intermediate lobe (IL) of adult male mouse. We revealed novel pituicyte markers showing higher specificity than previously reported. Single molecule in situ hybridization revealed spatial organization of the major cell types implying intercellular communications. We present a comprehensive molecular and cellular characterization of neurohypophyseal cell-types serving as a valuable resource for further functional research.Significance StatementThe neurohypophysis (NH) is a major neuroendocrine interface, which allows the brain to regulate the function of peripheral organs in response to specific physiological demands. Despite its importance, a comprehensive molecular description of cell identities in the NH is still lacking. Utilizing single cell RNA sequencing technology, we identified the transcriptomes of five major neurohypophyseal cell types in the adult male mice and mapped the spatial distribution of selected cell types in situ. We revealed an unexpected cellular heterogeneity of the neurohypophysis and provide novel molecular markers for neurohypophyseal cell types with higher specificity than previously reported.

Equine hyperadrenocorticism: A report of 4 natural cases

Hyperadrenocorticism, the most common equine endocrinopathy, is the result of hyperplasia or a functional adenoma of the pars intermedia (intermediate lobe) of the pituitary gland. The study population included a 20-year old Irish hunting gelding (case 1), a 13-year old English Thoroughbred stud horse (case 2), an 18-year old intact male donkey (case 3) and a 22-year old mare belonging to a mixed breed (case 4). From a historical and clinical standpoint, the most common clinical signs were hypertrichosis (4/4), progressive loss of body weight (3/4), polyuria-polydipsia (3/4), laminitis (3/4), depression-lethargy (2/4), sway-back (lordosis) (2/4), pot-belly (2/4), bulging of the supraorbital region due to the excessive deposition of adipose tissue (2/4), hyperhidrosis (2/4) and delayed wound healing (2/4). The results of the routine laboratory testing showed lymphopenia (4/4), anemia (2/4), mature neutrophilia (1/4), monocytosis (1/4), hypertriglyceridemia (4/4), increased ALP activity (3/4), hypocalcemia (3/4) and hypoalbuminemia (2/4). Also, case 2 developed type III diabetes mellitus as a consequence of the disease. The diagnosis of hyperadrenocorticism, that was initially based on the classical clinical signs, noticed in all 4 animals, was further confirmed in 3 of them (cases 1, 3, 4) with the overnight dexamethasone suppression test. Oral bromocryptine (dopamine agonist), that was subsequently prescribed in two horses (cases 1, 4) resulted in a remarkable clinical improvement, first noticed in both animals approximately two months after the beginning of the treatment.