Zona Reticularis Research Articles

SAT267 BMP4, GDF11 And TGFβ2 Repress Adrenal Cell Cortisol Production By Blocking CYP17A1 And CYP11B1 Expression

Abstract Disclosure: E.C. Fischbach: None. A. Lopez: None. M. Ullenbruch: None. J. Rege: None. W.E. Rainey: None. Introduction: The adrenal cortex is responsible for the biosynthesis of multiple steroid hormones that regulate electrolyte and carbohydrate balance. Cortisol, the principal glucocorticoid in humans, is primarily produced in the middle zone of the cortex known as the zona fasciculata (ZF). The enzymes responsible for cortisol biosynthesis include cytochrome P450c17 (17α-hydroxylase), encoded by CYP17A1 and cytochrome P450c11 (11β-hydroxylase), encoded by CYP11B1. Several paracrine/autocrine regulators, including members of the transforming growth factor beta (TGFβ) superfamily, have been implicated in controlling adrenocortical cell homeostasis and steroid hormone production. Previously, we reported a bone morphogenetic protein 4 (BMP4) expression gradient in the adrenal cortex with higher levels in the zona glomerulosa (ZG) and outer ZF compared to the zona reticularis (ZR). In this study, we sought to compare adrenal expression of members of the TGFβ family including BMP4, growth differentiation factor 11 (GDF11), and transforming growth factor beta 2 (TGFβ2), and determine their effect on adrenal cell cortisol biosynthesis. Methods: Growth factor mRNA levels were studied using microarray analysis of laser captured ZG, ZF and ZR from human adrenals. The adrenal cell line, HAC15 was used to determine the effects of the TGFβ family members alone and in combination. Output measures included cell cortisol production and mRNA levels of steroidogenic enzymes using ELISA and quantitative RT-PCR, respectively. Results: Microarray analysis indicated higher mRNA levels of BMP4, GDF11 and TGFβ2 in the ZG compared with the ZF and ZR regions of the adrenal. Treatment of HAC15 cells with either BMP4 (50 ng/mL), GDF11 (50 ng/mL) or TGFβ2 (10 ng/mL) significantly inhibited cortisol production, as well as CYP11B1 and CYP17A1 mRNA levels. The inhibition was observed under basal and forskolin-stimulated conditions. Furthermore, the inhibitory effects on cortisol production and enzyme expression were additive when factors were added together. Summary: Overall, our findings suggest that the human adrenal has a gradient of multiple members of the TGFβ family. The additive action of these growth factors on adrenal cell differentiation may explain, in part, the inability of the ZG to produce cortisol and its low expression of CYP11B1 and CYP17A1. Presentation: Saturday, June 17, 2023

FRI168 LH-hCG Receptor Is Expressed In All Three Zones Of Normal Adrenal Glands But Not In Aldosterone-Producing Micronodules

Abstract Disclosure: S.A. Reda: None. A. Kirschenbaum: None. S. Yao: None. L. Milgram: None. J. Rege: None. A.C. Levine: None. Embryologically, the adrenals and gonads develop from the urogenital ridge with differential migration and differentiation. Post-natally, pituitary adrenocorticotropic hormone (ACTH) stimulates adrenal cortical cell growth and steroidogenesis, whereas pituitary luteinizing hormone (LH) regulates gonadal cell growth and steroidogenesis. However, there are multiple reports of gonadal tumorigenesis driven by high ACTH (in congenital adrenal hyperplasia) and adrenal cortical tumorigenesis driven by high LH/human chorionic gonadotropin (hCG) levels (adrenal tumors in pregnant and postmenopausal females). We studied the immunohistochemical expression of LH/hCG receptor (LH/hCG-R) in normal adrenal glands from 12 kidney donors (four male and eight female, two of which were <50 years of age and six were >50 years of age). Some of these specimens also contained aldosterone producing micronodules (APMs). Staining was performed for Synaptophysin, aldosterone synthase (CYP11B2), LH/hCG-R, WNT/β-Catenin, and DLK-1. In normal adrenal tissue, we first used synaptophysin stain to identify the nerves in the adrenal cortex as well as the adrenal medulla (M). CYP11B2 staining demonstrated immunoreactivity in a linear pattern throughout the Zona Glomerulosa (ZG) in five of the 12 samples. Faint expression of LH/hCG-R was detected in all twelve samples throughout the three layers of the adrenal cortex: the ZG, Zona Fasciculata (ZF) and Zona Reticularis (ZR). However, there were occasional cells in both the ZR and ZF that stained intensely for LH/hCG-R expression. LH/hCG-R immunoreactivity was also noted in the progenitor-rich subcapsular (SC) region. WNT β-Catenin, a transcription factor involved in the proliferation of adrenal cortical cells, stained most heavily in the SC/ZG region, possibly representing adrenal cortical progenitor cells. DLK-1, an adrenal cortical progenitor cell marker, was also expressed in the subcapsular zone. In adrenal tissues containing APMs, CYP11B2 immunostaining revealed areas of CYP11B2 positive cells that dip into the ZF, with no preserved linear staining for CYP11B2 in the ZG. LH/hCG-R stained most densely in the SC and ZG regions, with little to no immunoreactivity in the APMs. WNT β-Catenin likewise stained the SC and ZG regions with no expression in the APMs. Our findings suggests that LH/hCG-R is expressed sporadically in normal adrenal cortical tissue, but densely at times in all three zones of the adrenal cortex and the subcapsular area where the putative adult progenitor cells reside. The LH/hCG-R does not appear to be expressed throughout the entirety of APMs. Presentation: Friday, June 16, 2023

OR08-03 Ectopic Localization Of CYP11B1 and CYP11B2-Expressing Cells In The Normal Human Adrenal Gland

Abstract Disclosure: C. Duparc: None. P. Camponova: None. M. Roy: None. H. Lefebvre: None. M. Thomas: None. The zonation of the human adrenal cortex has long been established morphologically and histologically as three distinct layers of cells. The outer zona glomerulosa (ZG) comprises densely packed cells arranged in clusters that produce aldosterone; the zona fasciculata (ZF) is composed of cells with large cytoplasm, containing lipid droplets arranged in radial columns that synthetize cortisol; and the zona reticularis (ZR) is composed of compact and pigmented cells producing androgens. The sharp line of demarcation between ZG and ZF has been recently challenged suggesting that this interface is no longer a compartment boundary. We have used immunohistochemical analyses to study the steroid 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) pattern of expression and investigate the remodeling of the adrenal cortex in relation to aging. We analyzed human adrenal glands prepared from 47 kidney donors (18 females, 29 males, ranging in age from 22 to 81 years old). No aldosterone-producing micronodules (APMs) were detectable in the younger donors aged between 22-39 but the functional ZG depicted by positive CYP11B2 staining demonstrated a lack of continuity. In contrast, the development of APMs was found in samples from individuals aged 40-70. None of the 47 adrenals studied had a continuous CYP11B2 expression which appeared to be fragmented within the subcapsular region. Importantly, the progressive replacement of CYP11B2-expressing cells in the histological ZG by CYP11B1-expressing cells highlights the remodeling capacity of the adrenal cortex. Cells expressing respectively CYP11B1 and CYP11B2 were found interwoven at different levels: i) single or cluster of CYP11B2 cells can be observed in the inner cortex surrounded by CYP11B1-expressing cells (33/47, 70.21%) and ii) the two type of cells might be found in the same nest or basket of cells beneath the capsule (40/47 donors, 85.10%). Interestingly, CYP11B1 and CYP11B2 cells were also present within the ZR. Proliferation detected by Ki-67 expression occurred in 14.04% (±1.69%) of the CYP11B2-expressing cells while it appeared expressed in 85.96% (±1.69%) of the CYP11B1-positive cells (p<0.001). No significant correlation between Ki-67 and age or sex has been found. Our data emphasize that mineralocorticoid- and glucocorticoid-producing cells are distributed throughout the cortex and the medulla making the determination of the functional status of a cell or group of cells a unique tool in deciphering the changes occurring in adrenal gland particularly during aging. Despite the fact that our data cannot exclude the existence of the CYP11B2+ cell conversion/migration lineage as demonstrated in mice they suggest that alternative mechanisms may exist in the human adrenal such as inward displacement of CYP11B2 expressing cells without lineage conversion. Presentation: Friday, June 16, 2023

OR27-05 Two-pronged Mediation Of Adrenal Steroidogenesis By Oxytocin

Abstract Disclosure: U. Cheliadinova: None. G. Pevnev: None. V. Ryu: None. T. Frolinger: None. S.L. Sims: None. M. Ofer: None. F. Korkmaz: None. O. Barak: None. J. Gimenez Roig: None. F. Sultana: None. N. Kramskiy: None. S. Wizman: None. M. Orloff: None. T. Yuen: None. D. Lizneva: None. M. Zaidi: None. A. Gumerova: None. Stress–stimulated glucocorticoid release affects diverse biological processes, including pregnancy and childbirth. The controversy surrounding effects of OXT on stress prompted us to investigate its actions on adrenal cortical function in mice. RNAscope and immunohistochemistry provided unequivocal evidence for abundant OXT receptor (OXTR) expression in the adrenal cortex, predominantly in zona fasciculata and zona reticularis. Female mice displayed higher adrenal cortical Oxtr expression, more than a range of other tissues under study. Oxtr expression was also detected in the adenohypophysis solely in female mice, while both male and female mice expressed Oxtr in the pars intermedia of the hypophysis. Aging impressively attenuated Oxtr expression in female mice in both the adrenal gland and adenohypophysis. For loss–of–function studies, we generated tissue–specific Star-CreERT2/Oxtrfl/fl mice in which the Oxtr was deleted from steroidogenic tissues, namely adrenals and ovaries. Deletion was confirmed by RNAscope and immunohistochemistry. We found no difference in serum corticosterone between tamoxifen–induced and uninduced mice; however, the elevation of serum corticosterone upon ACTH stimulation or after four weeks of stress was attenuated in tamoxifen–induced mice. Bulk RNAseq of adrenals from tamoxifen–induced mice revealed the downregulation of key steroidogenic genes, namely Cyp17a1, Hsd3b3, Hsd17b2, Cyp3a41b, and Serpina6 (a corticosteroid–binding globulin). In gain–of–function studies, a single OXT injection led to a rise of serum ACTH in tamoxifen–induced mice, which was greater in magnitude than uninduced controls. However, after five days of OXT injections, corticosterone levels became elevated, whereas ACTH levels declined to baseline. This suggests that OXT acts directly on OXTRs on corticotropes to initiate a stress–like response, with a negative feedback loop that inhibits further ACTH secretion. Of note is that male mice did not display any difference between tamoxifen–induced and uninduced groups, consistent with their low hypophyseal Oxtr expression. In all, we describe a novel gender–specific two–pronged circuit through which OXT may precisely regulates the production of corticosterone from the adrenal cortex. In the first arm, OXT directly stimulates ACTH secretion through adenohypophysis OXTRs. In the second arm, OXT prevents excessive stress–induced adrenal stimulation by downregulating steroidogenic genes. Our discovery lays the foundation for the emergence of new physiology that may relate to a fundamental role for oxytocin in the stress response during procreation, notably, when levels are elevated during pregnancy and lactation. Presentation: Saturday, June 17, 2023

FRI166 Characterization of Aldosterone-producing Cell Cluster (APCC) At Single-cell Resolution

Abstract Disclosure: N. Iwahashi: None. H. Umakoshi: None. T. Seki: None. C. Gomez-Sanchez: None. K. Mukai: None. M. Suematsu: None. Y. Umezawa: None. M. Oya: None. T. Kosaka: None. M. Seki: None. Y. Suzuki: None. Y. Horiuchi: None. K. Nishimoto: None. Y. Ogawa: None. Context: The adrenal cortex consists of zona glomerulosa (ZG), fasciculata (ZF), and reticularis. Aldosterone-producing cell clusters (APCCs) that express aldosterone synthase (CYP11B2) strongly can be frequently found in adult adrenals and harbor somatic mutations that are also present in aldosterone-producing adenomas (APAs). Primary aldosteronism is mainly caused by APAs or idiopathic hyperaldosteronism (IHA). We presume that APCCs are causing IHA and are precursors of APAs. However, the gene expression characteristics and especially the development of APCCs are not well understood. Objective: This study aimed to analyze the transcriptome of APCCs at single-cell resolution and infer the developmental trajectory. Methods: Single-cell RNA sequencing (scRNA-seq) was performed on two adult adrenals. Results: Immunohistochemical analyses confirmed the presence of APCCs in the two adrenals. scRNA-seq data of 6770 adrenal cells were obtained and 2928 high-quality cells were selected after quality control procedures. Unsupervised clustering and marker gene expressions such as CYP11A1 and NR5A1 identified 1765 adrenocortical cells, which were further divided into six clusters (B1-6). Using the list of APCC/ZG upregulated genes from our previously reported DNA microarray study, three clusters (B1, B3, and B6, totaling 923 cells) were identified as APCC/ZG cells. By further subclustering, the APCC/ZG cells were divided into 3 clusters (C1, C2, and C3). The APCC cluster (C3, 64 cells) and ZG cluster (C1, 447 cells) were identified. Cluster C2 seemed to be ZG-to-ZF transitional cells. RNA velocity analysis, which aimed to infer the direction of differentiation, inferred a direction of development from ZG cells to APCCs. Conclusion: Our results revealed the gene expression characteristics of APCC at single-cell resolution and show that some ZG cells remodel into APCCs. Presentation: Friday, June 16, 2023

OR08-02 In Situ Reconstruction Of Distinct Normal And Pathological Cell Populations Within The Human Adrenal Gland Using Spatial Transcriptomics

Abstract Disclosure: F. Rui: None. K. Walters: None. K. Koc: None. A. Baldwin: Stock Owner; Self; Tempus Labs. K. Kiseljak-Vassiliades: Advisory Board Member; Self; HRA Pharma. L.M. Fishbein: Consulting Fee; Self; Lantheus/Progenics Azedra. N. Mukherjee: None. The human adrenal gland is a vital endocrine organ controlling a multitude of functions through the secretion of cortical steroid and medullary catecholamine hormones. Several outstanding questions remain about the adrenal spatial organization and related differential developmental signaling pathways which have functional consequences within and between the cortex and medulla. The current model of the functional zonation of the cortex posits a centripetal differentiation process in which the cortical cells arise in the capsule and migrate inwards changing their gene expression patterns as they progress to the three different functional layers of the cortex. The evidence for this model is based on mouse lineage tracing experiments, and it is unclear to what extent it applies to human adrenal glands. To address this, we performed spatially resolved transcriptomics on four adult adrenal sections from two different human specimens to identify cell populations and their spatial relationship in this highly organized and functionally diverse organ. We identified 14 distinct cell populations; 79% of these spots represented the cortex or medulla and exhibited the concentric expression pattern reflecting the known spatial organization of the human adrenal gland. Trajectory inference analysis of the cortical cells showed continuous expression transitions from cells of the capsule to zona glomerulosa to zona fasciculata to zona reticularis. These data demonstrate for the first time that the centripetal differentiation model for functional zonation in mice is consistent for adult human adrenal tissue. We also validated the spatial expression pattern of a subset of WNT-related genes specifically expressed with distinct patterns in the capsule and zona glomerulosa cells. These genes represent novel regulatory factors that may be crucial in the differentiation of adult human adrenal cortex. Finally, we observed two CYP11B2 positive aldosterone producing cell clusters (APCC) in the adrenal tissue section from a 72-year-old woman. APCCs are thought to play a role in the pathological progression of primary aldosteronism; however, it is unclear if there are unifying expression signatures other than CYP11B2 protein expression. Our analysis found both similarities and differences to previously published data sets of APCC signatures. It is unclear to what extent these expression differences are technical (i.e. different platforms) or due to biological heterogeneity (between individuals or APCCs). The subgroup of similar gene expression in APCCs across all studies thus far may represent a core APCC signature which should be validated on a larger cohort. Altogether, our study uses spatial transcriptomics of the normal human adrenal and the results have important implications for the differentiation and functional zonation,as well as, for the early dysregulation leading to primary aldosteronism. Presentation: Friday, June 16, 2023

Changes in the microscopic organisation of rat adrenal glands under the influence of Vipera berus berus venom

Snakebite envenoming is a common but neglected public health problem worldwide. Annual mortality as a result of snakebites exceeds 138,000. The organs of the endocrine system are among the first to react to the effects of snake and viper toxins. Under these conditions, the adrenal glands are involved in the pathological process and contribute to the formation of the adaptation syndrome, undergoing complex structural changes. The research aims to study the changes in the microscopic organization of the adrenal glands of rats under the influence of Vipera berus berus venom. Experimental studies were carried out on white non-linear male rats. The animals were conditionally divided into a control and an experimental group of 10 individuals. Experimental rats were injected intraperitoneally in a saline solution with a semi-lethal dose (LD50) (1.576 mg/g-1) of Vipera berus berus venom. Animals of the control group were injected intraperitoneally with only saline solution. Rats were removed from the experiment 24 hours after exposure to the venom and anaesthetized by decapitation. Adrenal gland samples were taken for microscopic examination. Fixation of the material and preparation of paraffin blocks were carried out according to generally accepted methods. Histological preparations of the adrenal glands were stained with hematoxylin and eosin. Histological preparations were studied using an SEO SСAN light microscope. Under the influence of Vipera berus berus venom in zona glomerulosa of the adrenal cortex, moderately pronounced pathological changes were found, including vacuolization and granularity of the cytoplasm of endocrinocytes, loss of precise contours of nuclei, their hyperchromasia, expansion of lumens of sinusoidal capillaries, accumulation of erythrocytes in them. Under these conditions, zona fasciculata is characterized by significant cell granularity and perinuclear edema. Less pronounced structural organization changes were noted in the zona reticularis of the adrenal cortex. Endocrinocytes of this zone had small sizes, eosinophilic cytoplasm and dark nuclei. In the medulla of the adrenal glands, the cells were large in size and had indistinct contours, the cytoplasm was characterized by basophilic granularity, and the nuclei were light due to the predominance of euchromatin. The most pronounced effect of Vipera berus berus venom was on the zona glomerulosa and zona fasciculata of the adrenal cortex; most of the morphological signs of pathology in which were caused by a violation of protein metabolism in the cells of the parenchymal and stromal elements of this organ.

Age Related Histoarchitectural Studies on the Adrenal Gland of Goat (Capra hircus)

Background: Adrenal gland is an important endocrine gland that secrets several hormones that play a key role in the adaptation of the newborn to extra uterine life. The study was conducted to explore the histomorphological development of adrenal glands in Gohilwadi Goat (Capra hircus). Methods: The research work was carried out on twenty pair of goat adrenal gland collected from local abattoir. The animals were divided into three age groups, viz. group- I (day old to 1 month), group- II ( greater than 1 month to 6 months) and group- III ( greater than 6 months). Result: Histological observations revealed that the gland was fully differentiated into cortex and medulla, completely surrounded by a dense fibrous capsule in the day old goats. In group- I, adrenal cortex was made up of definite and foetal cortex, whereas former one comprised of zona glomerulosa and zona fasciculata only. Small to elongate foetal cortical cells were characterized by eosinophilic cytoplasm and arranged into irregular cords. In group II, the foetal cortex disappeared and only definite cortex was present with well-developed three zones i.e. Zona glomerulosa, zona fasciculata and Zona reticularis. Zona glomerulosa was the smallest of the three cortical zones and thickness increased from group-I to group-III. The zona fasciculata was the largest among the three cortical zones in all the age groups and thickness increased from group-I to group-III. Zona reticularis was well organized in the adrenal gland of adult Gohilwadi goat. Medulla occupied greater proportion of the gland and inner medullary cells were the main cellular constituent of medulla. Thickness of medulla increased from group-I to group-III.

Primary Aldosteronism: Spatial Multiomics Mapping of Genotype-Dependent Heterogeneity and Tumor Expansion of Aldosterone-Producing Adenomas.

Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

THE CORRELATION OF REACTIVE CHANGES IN THE CORTICAL AND CEREBRAL SUBSTANCE OF THE ADRENAL GLANDS OF WHITE RATS DURING THE CORRECTION OF TESTOSTERONE BLOCKADE AT THE 6TH MONTH OF THE EXPERIMENT

The main pharmacological effect of prolonged injection of a synthetic analogue of gonadotropin-releasing hormone is the development of chemical castration. To correct the reactive changes that develop during testosterone blockade in the organs under study, scientists use the active ingredient quercetin to study its effect on oxidative stress. Its antioxidant effect can be used for pathogenetic therapy and prevention of changes in the body caused by a decrease in testosterone levels [12, 13, 18]. The aim of the study was to investigate morphological changes in corticosterocytes zones of the cortex and medullary endocrinocytes of the adrenal glands of white rats during the correction of central testosterone deprivation at the sixth month of the experiment. Materials and methods: The adrenal glands of 25 male white rats were studied and divided into 3 groups: group 1 – 5 intact rats, group 2 – 10 rats injected with triptorelin acetate subcutaneously at a dose of 0.3 mg of active substance per kg of body weight, and group 3 – 10 rats injected with quercetin 100 mg per kg of body weight once a day 3 times a week for 6 months against the background of triptorelin acetate administration. The average size and volume of the nucleus (N) of corticosterocytes of all cortical zones (CZ), the average size and volume of the nucleus of the ChC, the density of the cords of the adrenal cortex zones and the density of the cords of the adrenal medulla were measured and calculated. Conclusions: 1. Correction of central testosterone deprivation at the 6th month of the experiment causes a decrease in the synthetic activity of corticosterocytes of the glomerular zone and an increase in the protein synthetic function of fascicular zone cells. 2. A significant decrease in the average size of corticosterocytes of the reticular zone and restoration of cytoplasmic basophilia at 6 months of correction proves the positive effect of quercetin on the synthetic activity of these cells. 3. Reduction of the average size and volume of chromophilic endocrinocyte nuclei and increase in their number during the correction of chemical castration at 6 months of the experiment confirms the significant positive effect of quercetin. 4. The revealed correlation between changes in the cells of the glomerular, fascicular and reticular zones of the adrenal cortex and the adrenal medulla of white rats against the background of chemical castration and its correction indicates the involvement of not only hypothalamic-pituitary dependent endocrine organs, but also organs of the sympathoadrenal system.

Вплив наночастинок золота на морфофункціональний стан гіпоталамуса та кори надниркових залоз щурів при ожирінні

The aim of our study was to compare the effects of a gold salt (sodium tetrachloroaurate) and gold nanoparticles colloidal solution on the morphofunctional state of the hypothalamus and adrenal glands of 6-month-old male rats with obesity, which was induced using a diet-induced model of metabolic disorders. The rats, modeled for obesity, were divided into three groups that received a NaCl solution, gold salt solution and colloidal solution of gold nanoparticles for 10 days. Rats of the control group received a standard diet. After the end of the experiment, the hypothalamic nuclei and adrenal glands, prepared according to the appropriate histological methods, were collected from the animals. In the paraventricular nucleus of the hypothalamus, a small-cell area of neurons was examined. In the adrenal glands, cells of three zones of the cortex were examined: glomerular, fascicular, and reticular. The results of the study showed that obesity led to the increase of functional activity in the nuclei of neurons in the small-cell region of the paraventricular nucleus of the rat hypothalamus. Functional activity increased maximally in the cells of the glomerular zone of the adrenal cortex, moderately increased in the fascicular zone, and no significant changes were recorded in the reticular zone. It was shown that the gold salt caused a decrease in the functional activity of neurons of the paraventricular nucleus of the hypothalamus in obese rats. In the glomerular zone of the adrenal cortex, the functional activity of cells significantly decreased; in the fascicular zone, the parameters of cell nuclei decreased to a greater extent; in the reticular zone, there were almost no changes. The introduction of gold nanoparticles colloidal solution caused the intensification of functional activity of the paraventricular hypothalamic nucleus cells in obese rats. In the glomerular zone of the adrenal cortex, cell parameters decreased; in the fascicular zone, cytoplasmic parameters decreased, but nuclear parameters did not change; in the reticular zone, measured values of nuclei increased and cytoplasmic parameters remained unchanged. In general, the administration of gold nanoparticles colloidal solution produced more pronounced effects than gold salt solution on cells of the paraventricular nucleus of the hypothalamus and the adrenal cortex in obese rats.

Parabrachial tachykinin1-expressing neurons involved in state-dependent breathing control

Breathing is regulated automatically by neural circuits in the medulla to maintain homeostasis, but breathing is also modified by behavior and emotion. Mice have rapid breathing patterns that are unique to the awake state and distinct from those driven by automatic reflexes. Activation of medullary neurons that control automatic breathing does not reproduce these rapid breathing patterns. By manipulating transcriptionally defined neurons in the parabrachial nucleus, we identify a subset of neurons that express the Tac1, but not Calca, gene that exerts potent and precise conditional control of breathing in the awake, but not anesthetized, state via projections to the ventral intermediate reticular zone of the medulla. Activating these neurons drives breathing to frequencies that match the physiological maximum through mechanisms that differ from those that underlie the automatic control of breathing. We postulate that this circuit is important for the integration of breathing with state-dependent behaviors and emotions.

Accelerated telomere shortening in adrenal zona reticularis in patients with prolonged critical illness.

The number of patients with prolonged critical illness (PCI) has been increasing in many countries, and the adrenal gland plays an important role in maintaining homeostasis during PCI. Chronic disease burden is reportedly associated with shorter telomere lengths in human tissues. Telomere shortening in human somatic cells is largely dependent on cell divisions, and critically short telomeres lead to cellular dysfunction and aging. However, the association between PCI and telomere lengths in human adrenal cells is poorly understood. In this study, we investigated this association to assess whether the burden of PCI could accelerate the aging process in adrenal cells. Adrenocortical tissues from patients who died after PCI usually show a diffuse pattern of intracellular cholesterol ester depletion (i.e., lipid depletion). This study examined near-normal adrenal glands obtained from autopsied patients who died suddenly (control group) and lipid-depleted adrenal glands obtained from autopsied patients who died after PCI (PCI group). The control group included 7 men aged 80 to 94 years (mean age: 85.3 years) and 7 women aged 84 to 94 years (mean age: 87.7 years). The PCI group included 10 men aged 71 to 88 years (mean age: 78.8 years) and 8 women aged 77 to 95 years (mean age: 85.6 years). By using quantitative fluorescence in situ hybridization, relative telomere lengths (RTLs) were determined in the parenchymal cells of the three adrenocortical zones (zona glomerulosa, zona fasciculata, and zona reticularis [ZR]) and in the chromaffin cells of the medulla. The number of adrenal parenchymal cells was determined by immunohistochemistry and digital image analysis. RTLs in ZR cells were significantly shorter in the PCI group than in the control group for both men and women (P = 0.0001 for men and P = 0.0012 for women). However, RTLs in the remaining three types of adrenal cells did not differ between the control and PCI groups for both men and women. The number of ZR cells was higher in the PCI group than in the control group for both men and women (P < 0.0001 for both men and women). The proportion of the number of ZR cells to the total number of adrenocortical parenchymal cells was also higher in the PCI group than in the control group (P < 0.0001 for both men and women). The Ki-67 proliferation index in ZR cells was higher in the PCI group than in the control group (P = 0.0039 for men and P = 0.0063 for women). This study demonstrated ZR cell-specific telomere shortening in patients with adrenal lipid depletion who died after PCI. Our results suggest that the reactive proliferation of ZR cells accelerates the telomere shortening and aging process in ZR cells in these patients. The results of our study may contribute to the understanding of adrenal aging during PCI.

Влияние введения мексидола на фоне 60-дневного воздействия тартразина на ультраструктуру эндокриноцитов коркового вещества надпочечных желёз крыс

Methods. In the experiment, 30 white mature rats were divided into 5 groups: Group 1 — control, Group 2 and Group 3 — rats exposed to tartrazine at the rate of 750 and 1500 mg/kg for 60 days. In the Group 4 and Group 5, mexidol was injected intramuscularly at the rate of 50 mg/kg against the background of the conditions of the Group 2 and Group 3. Electron microscopic examination of the adrenal glands was performed according to the standard protocol. Results. In the Group 2, epithelial cells of the glomerular and fascicular zones have a round or oval nucleus with uneven contours. The nucleus contains accumulations of heterochromatin under the karyolemma and in the karyoplasm. Large mitochondria with vesicular cristae are observed in the cytoplasm. The cisterns of the smooth endoplasmic reticulum are poorly developed. Lipid inclusions are located in groups or singly. In the Group 3, mitochondria with partially destroyed cristae were detected in the cytoplasm of endocrinocytes of the zona glomerulosa, and in the epithelial cells of the zona fasciculata, loosely packed lamellar structures were detected in the center or periphery of mitochondria, as well as cells with apoptosis. Endocrinocytes of the zona reticularis have a small size, an oval nucleus with slightly tortuous contours and accumulations of heterochromatin in the karyoplasm in the rats of the Group 2. Mitochondria were large and medium in size with spherical cristae in cross section, the cisterns of the smooth endoplasmic reticulum are unevenly expanded and fill the gaps between mitochondria. In the Group 3, almost half of the large mitochondria have densely packed lamellar structures along the periphery or in the center. Conclusion. The above-established ultramicroscopic features of endocrinocytes of the adrenal cortex after 60 days of exposure to tartrazine indicate a decrease in their secretory activity, which depends on the dose of the supplement administered. The introduction of mexidol against this background helps to restore the disturbed ultramicroscopic picture of cells to control values, which is more pronounced in the Group 4.

Ectopic localization of CYP11B1 and CYP11B2-expressing cells in the normal human adrenal gland.

The sharp line of demarcation between zona glomerulosa (ZG) and zona fasciculata (ZF) has been recently challenged suggesting that this interface is no longer a compartment boundary. We have used immunohistochemical analyses to study the steroid 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) pattern of expression and investigate the remodeling of the adrenal cortex in relation to aging. We analyzed human adrenal glands prepared from 47 kidney donors. No aldosterone-producing micronodules (APMs) were detectable in the younger donors aged between 22-39 but the functional ZG depicted by positive CYP11B2 staining demonstrated a lack of continuity. In contrast, the development of APMs was found in samples from individuals aged 40-70. Importantly, the progressive replacement of CYP11B2-expressing cells in the histological ZG by CYP11B1-expressing cells highlights the remodeling capacity of the adrenal cortex. In 70% of our samples, immunofluorescence studies revealed the presence of isolated or clusters of CYP11B2 positive cells in the ZF and zona reticularis. Our data emphasize that mineralocorticoid- and glucocorticoid-producing cells are distributed throughout the cortex and the medulla making the determination of the functional status of a cell or group of cells a unique tool in deciphering the changes occurring in adrenal gland particularly during aging. They also suggest that, in humans, steroidogenic cell phenotype defined by function is a stable feature and thus, the functional zonation might be not solely maintained by cell lineage conversion/migration.

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

Effect of Nonylphenol on the Structure of Adrenal Cortex in F1 Generation Rats

Previous studies, including our own, indicate that distinct morphological changes in rodent adrenal cortex could be induced by exposure of endocrine disrupting chemicals (EDC). In the present study, we conducted histological analyses of adrenocortical substructure using a nonylphenol-treated F1 rat model. The adrenal weight of NP-5000 group was significantly declined in female rats (p<0.001), while the adrenal weights of NP-treated groups were not significantly changed in male rats. The thickness of zona glomerulosa layers of female rats in NP-5000 group was significantly declined (p<0.001) but zona fasciculata layers were not changed. The zona reticularis layers of NP-treated group were significantly thinner than those of control group (NP-50, p<0.05; NP-5000, p<0.01). In male adrenal glands, there was no significant change of zona glomerulosa layers in NP-treated groups while the thickness of zona fasciculata in NP-5000 group was significantly decreased (p<0.01). Like female rats, the thickness of zona reticularis in NP-treated groups was significantly decreased (NP-50, p<0.001; NP-5000, p<0.05). Present study demonstrated that the adrenal histology could be altered by low-dose NP exposure in F1 rats, and the effect might be sexually dimorphic. Further study will be helpful for understanding possible adrenal pathophysiology induced by EDC exposure, and EDC-related sexually dimorphic phenomena in rodent adrenals.

COMPARATIVE CHARACTERISTICS OF CORD DENSITY IN FASCICULAR AND RETICULAR ZONES OF ADRENAL GLANDS IN WHITE RATS UNDER CENTRAL TESTOSTERONE BLOCKADE

Introduction. Gonadotropin-releasing hormone has been proven to act as an important regulator of the hypothalamic-pituitary-gonadal axis. Analogues of its agonists and antagonists are effective in the treatment of diseases of the male and female reproductive systems. Clinicians pay special attention to the study of the activity of biochemical regulators in the structures of the hypothalamic-pituitary-adrenocortical system under the administration of different medications, for instance, methanandamide, dapagliflozin, triptorelin acetate, and others. Knowing reactive changes in the fascicular and reticular zones of the adrenal glands under central testosterone deprivation by administering "Diferelin", a synthetic analogue of gonadotropin-releasing hormone enables to demonstrate the relationship between the male gonads, cortical zones of the adrenal glands and hypothalamus at the microscopic level. The purpose of this study is to investigate the density of cords in the fascicular and reticular zones of the adrenal glands of white rats under central testosterone blockade in 1, 3, 6, and 9 month periods of the experiment.&#x0D; Materials and methods. The study included 50 white male rats divided into 5 groups: 1 group included 10 intact rats and 2, 3, 4, 5 groups (10 test animals in each) were injected with the "Diferelin". Adrenal tissues were taken at the end of 1, 3, 6 and 9 months of the experiment.&#x0D; Results and discussion. For the entire duration of the experiment the quantitative composition of corticosterocytes in the studied areas of the fascicular zone (FZ) and reticular zone (RZ) reflected the density index (DI) of the cords of the studied zones in the area with average index of 36422.511±2082.09 μm².&#x0D; Morphometric study of the density of the adrenal medulla and cortical substance enabled to identify the main terms of the development of reactive changes in the adrenal glands under central testosterone deprivation.&#x0D; Conclusion. Increased reactivity of corticosterocytes in the fascicular zone from the 1st month of the experiment with the peak value at the 6th month proves the participation of this zone in the stress response under central testosterone deprivation.&#x0D; A significant increase in the parenchymal component of the reticular zone from 3 to 6 months indicates a compensatory activation of corticosteroids in response to testosterone blockade.

Изменения морфометрических показателей коркового вещества надпочечников у самок марала в течение года

The paper studies the histological structure of the adrenal cortex in non-pregnant sexually mature maral does in different seasons of the year (exemplified by animals living on the free-range farms of the Altai Republic). The greatest thickness of the adrenal cortex is observed during winter. The minimum thickness of the zona glomerulosa is typical of summer, while in autumn the cell size increases significantly. The zona fasciculata expands during winter, reaching minimum dimensions in spring. The minimum thickness of the zona reticularis is identified in winter; its cells and nuclei decrease statistically significantly in size during this season compared to autumn.

PSAT040 Bilateral Adrenal Infarcts: Initial Presentation in Concurrent Myelodysplastic Syndrome and Anti-Phospholipid Syndrome

Abstract Introduction Bilateral adrenal hemorrhage (BAH) has been noted in several conditions. Bilateral adrenal infarcts (BAI) are rarer but have been seen in anti-phospholipid syndrome (APS). Adrenal insults are not commonly associated with myelodysplastic syndrome (MDS). We present a patient who developed BAI as initial presentation of APS in the setting of MDS and discuss management considerations. Case A 58-year-old female with MDS diagnosed one month prior presented with abdominal pain, present for one year but acutely worse in the past week. CT A/P showed nodularity of bilateral adrenal glands. She was treated conservatively but continued to experience nausea, vomiting, and abdominal pain – repeat CT A/P one month later was suspicious for bilateral peri-adrenal hemorrhages. MRI with adrenal protocol demonstrated bilateral non-hemorrhagic adrenal infarcts. VS were negative for hypothermia or hypotension. Labs showed Na 136 (135-145 mmol/L), K 3.5 (3.4-5.1 mmol/L), BG 108 (mg/dL), DHEA-S 9.3 (8.0-391 mcg/dL), renin 6.7 (0.5-4.0 ng/mL/hr), aldosterone 6.2 (&amp;lt;31.0 ng/dL), 60-minute cortisol after ACTH stimulation test 34.3 (5.2-22.5 mcg/dL), plasma metanephrines &amp;lt;0.10 (0.00-0.49 nmol/L), plasma normetanephrines 0.34 (0.00-0.89 nmol/L), Hgb 11.8 (12.0-15.5 g/dL), and plts 129K (140-450 K/mcL). No history of anticoagulant use, trauma, or VTE. COVID-19 PCR was negative on two separate tests. Hypercoagulability testing noted positive lupus anticoagulant. Anticoagulation was deferred due to underlying bone marrow abnormalities and thrombocytopenia; aspirin was recommended instead by hematology. Outpatient testing revealed 12pm cortisol of 12.6 (5.2-22.5 mcg/dL), ACTH 108.3 (7.2-63.0 pg/mL), and DHEA-S 6.2 (8.0-391 mcg/dL). Patient was empirically started on hydrocortisone for glucocorticoid replacement therapy. Conclusion BAI is not typically associated with MDS but can occur in the setting of a concurrent hypercoagulable process such as APS. The adrenal glands are the most commonly involved glands in APS. Patients often present in adrenal crisis but can present in stable condition as in this case. The adrenal glands are particularly vulnerable due to the "vascular dam" near the zona reticularis. Imaging and laboratory investigation can clarify the nature of the adrenal insult and guide management. Treatment strategy may include anticoagulation to prevent further VTE events, but bleeding risk must be considered. Both glucocorticoid and mineralocorticoid replacement should be considered until residual adrenal reserve can be fully elucidated, which may take several months from initial diagnosis. References Rao RH. Bilateral massive adrenal hemorrhage. Med Clin North Am 1995; 79(1): 107-29. Aron DC, Findling JW, Tyrrell JB. Glucocorticoids and adrenal androgens. In: Greenspan's Basic and Clinical Endocrinology. Gardner DG, Shoback D (eds). Mc Graw Hill 2007; pp 367-378. Caron P, Chabannier MH, Cambus JP, Fortenfant F, Otal P, Suc JM. Definitive adrenal insufficiency due to bilateral adrenal hemorrhage and primary antiphospholipid syndrome. J Clin Endocrinol Metab 1998; 83(5): 1437-9. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.