Non-coding variation in disorders of sex development.

Genetic studies in Disorders of Sex Development (DSD), representing a wide spectrum of developmental or functional conditions of the gonad, have mainly been oriented towards the coding genome. Application of genomic technologies, such as whole-exome sequencing, result in a molecular genetic diagnosis in ∼50% of cases with DSD. Many of the genes mutated in DSD encode transcription factors such as SRY, SOX9, NR5A1, and FOXL2, characterized by a strictly regulated spatiotemporal expression. Hence, it can be hypothesized that at least part of the missing genetic variation in DSD can be explained by non-coding mutations in regulatory elements that alter gene expression, either by reduced, mis- or overexpression of their target genes. In addition, structural variations such as translocations, deletions, duplications or inversions can affect the normal chromatin conformation by different mechanisms. Here, we review non-coding defects in human DSD phenotypes and in animal models. The wide variety of non-coding defects found in DSD emphasizes that the regulatory landscape of known and to be discovered DSD genes has to be taken into consideration when investigating the molecular pathogenesis of DSD.

Identification of glucagon-producing cells (A cells) in dog gastric mucosa.

An immunocytochemical technique using specific antiglucagon serum reveals the presence of glucagon-containing cells situated exclusively in the oxyntic glandular mucosa of the dog stomach. Electron microscope examination of the mucosa demonstrated endocrine cells containing secretory granules with a round dense core surrounded by a clear halo, indistinguishable from secretory granules of pancreatic A cells. Like the alpha granules of pancreatic A cells, the granules of these gastric endocrine cells exhibited a peripheral distribution of silver grains after Grimelius silver staining. Moreover, the granules of these cells were found to be specifically labeled with reaction product, using the peroxidase immunocytochemical technique at the ultrastructural level. Accordingly, these cells were named gastric A cells. These data suggest that the gastric oxyntic mucosa contains cells indistinguishable cytologically, cytochemically, and immunocytochemically from pancreatic A cells. It is believed that gastric A cells are responsible for the secretion of the gastric glucagon.

Endocrine pancreas: three-dimensional reconstruction shows two types of islets of langerhans.

Three-dimensional reconstructions of islets of Langerhans, based on immunofluorescent staining of successive serial sections with antiserums to insulin, glucagon, somatostatin, and pancreatic polypeptide reveal a marked difference in the number of cells containing glucagon and pancreatic polypeptide depending on the anatomical location of the islet in the pancreas. The two types of islets are situated in regions of exocrine tissue that are drained by different excretory ducts. This demonstration contradicts the assumption that all islets in the pancreas are similar in their endocrine cell content.

Glucagon: role in the hyperglycemia of diabetes mellitus.

Glucagon suppression by somatostatin reduces or abolishes hyperglycemia in dogs made insulin-deficient by somatostatin, alloxan, or total pancreatectomy. This suggests that the development of severe diabetic hyperglycemia requires the presence of glucagon, whether secreted by pancreatic or newly identified gastrointestinal A cells, as well as a lack of insulin. Glucagon suppression could improve therapeutic glucoregulation in diabetes.

Identification of glucagon in the gastrointestinal tract.

Gel filtration studies on Bio-Gel P-10 columns of a 50-fold purified porcine duodenal extract revealed a main peak of glucagon-like immunoreactivity (GLI) in the 2,900 mol wt zone and a smaller peak in the 3,500 mol wt zone, the same zone as the pancreatic glucagon marker. Like pancreatic glucagon, samples of 3,500 mol wt material gave essentially identical measurements in radioimmunoassays employing the pancreatic glucagon-specific antiserum 30K and the GLI crossreacting antiserum 78J, whereas the 2,900 mol wt peptide gave 60-fold higher readings in the 78J assay. On disk gel electrophoresis, the 3,500 mol wt fraction, like pancreatic glucagon, migrated at pH 8.3, whereas the 2,900 mol wt peptide remained at the origin; at pH 4.7, the 2,900 mol wt peptide migrated while the 3,500 mol wt immunoreactive peptide and glucagon remained at the origin. Isoelectric focusing revealed the 3,500 mol wt moiety to have an isoelectric point (pI) of 6.2, the same as pancreatic glucagon, whereas the 2,900 mol wt peptide had an pI greater than 10. The glycogenolytic activity of the 3,500 mol wt peptide in the perfused rat liver did not differ significantly from glucagon, and its adenylate cyclase stimulating activity in partially purified liver cell membranes was comparable to that of glucagon; the 2,900 mol wt peptide had less than 20% of these activities. In samples of 3,500 mol wt material subjected to isoelectric focusing, adenylate cyclase-stimulating activity was confirmed to fractions containing 30K immunoreactivity with a pI of 6.2. In samples of 2,900 mol wt material subjected to isoelectric focusing, adenylate cyclase-stimulating activity was confined to fractions containing 78J immunoreactivity with an pI greater than 10. Displacement of [125-I]glucagon from the membranes was limited to these two biologically active fractions. However, the affinity of both pancreatic glucagon and the 3,500 mol wt peptide was an order of magnitude greater than of the 2,900 mol wt peptide. Thus, by all of several biologic, physiocochemical, and immunometric techniques, the 3,500 mol wt gut immunoreactive peptide could not be distinguished from pancreatic glucagon, while the 2,900 mol wt peptide was readily differentiated by all these techniques. "True" A-cells, ultrastructurally indistinguishable from pancreatic A-cells but differing from the A-like cells of the lower bowel, were identified in the gastric fundus of dogs. Their distribution corresponded to that of the 3,500 mol wt immunoreactivity resembling pancreatic glucagon, while the distribution of "A-like cells" in the lower small intestine corresponded to that of GLI.

Reduced beta-cell glucose transporter in new onset diabetic BB rats.

Previous studies from our laboratories have suggested a defect in glucose transport in islets isolated from BB rats on the first day of overt diabetes. To quantitate by immunostaining the glucose transporter of beta-cells (GLUT-2) before and at the onset of autoimmune diabetes we employed an antibody to its COOH-terminal octapeptide. On the first day of overt diabetes, defined as the day the daily blood glucose first reached 200 mg/dl, the volume density ratio of GLUT-2-positive to insulin-positive beta-cells was only 0.48 +/- 0.06, compared to 0.91 +/- 0.02 in age-matched nondiabetic diabetes-resistant controls (P less than 0.001). In age-matched nondiabetic diabetes-prone rats, most of which would have become diabetic, the ratio was 0.85 +/- 0.02, also less than the controls (P less than 0.05). Protein A-gold labeling of GLUT-2 in beta-cells of day 1 diabetic rats revealed 2.17 +/- 0.16 gold particles per micrometer length of microvillar plasma membranes compared to 3.91 +/- 0.14 in controls (P less than 0.001) and 2.87 +/- 0.24 in the nondiabetic diabetes-prone rats (P less than 0.02). Reduction in GLUT-2 correlates temporally with and may contribute to the loss of glucose-stimulated insulin secretion that precedes profound beta-cell depletion of autoimmune diabetes.

Morphologic and functional changes in sympathetic nerve relationships with pancreatic alpha-cells after destruction of beta-cells in rats.

Insulin-dependent diabetes mellitus (IDDM) in humans is accompanied by an attenuation of the response of glucagon to hypoglycemia. To identify an animal model of IDDM with alpha-cell unresponsiveness to glucopenia in which to pursue morphologic and in vitro functional investigation of the lesion, pancreases isolated from rats with IDDM induced by streptozocin (STZ) or occurring spontaneously in BB/W rats were perfused with buffer containing 150, 25, and 150 mg/dl of glucose. In both forms of IDDM the normal glucagon rise during glucopenia was markedly impaired, suggesting an abnormality comparable to that of human IDDM. Studies of the insular sympathetic apparatus were conducted in these rat models. Electron-microscopic examination of peri-insular nerve endings disclosed no discernible abnormality in either form of rat IDDM. However, morphometric analysis of contacts between [3H]norepinephrine-labeled sympathetic nerve terminals and alpha-cells in pancreases from STZ-induced diabetic (STZ-D) rats revealed a 65-70% reduction in direct contacts. An 80% reduction in the number of nerve endings (not labeled) in direct contact with alpha-cells was also noted in the BB/W diabetic rats. Norepinephrine reuptake, studied only in the STZ-D group, was not impaired. The availability of local endogenous norepinephrine to alpha-cells and their sensitivity to exogenous norepinephrine was determined by perfusing 2, 5, or 10 micrograms/ml of tyramine, a releaser of endogenous norepinephrine, and norepinephrine at a concentration that in pancreases from nondiabetic rats gave a quantitatively similar glucagon response.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of glucagonomas by light and electron microscopy and immunofluorescence.

Five tumors associated with the complete glucagonoma syndrome, as well as a series of glucagon-cell adenomas from three patients without this syndrome, were investigated by light and electron microscopy and by immunofluorescence. All tumors associated with the syndrome were large, from 3 to 35 cm along the major axis, and three of them were proved to be malignant. No common histologic arrangement of tumor cells was apparent for the five neoplasms examined. Immunofluorescent staining for glucagon and glicentin was carried out: while most cells were negative, a varying number of scattered cells were positive with both antisera in all tumors except one; three tumors contained more glicentin- than glucagon-immunoreactive cells. Moreover, three tumors were multihormonal, witn cells positive for pancreatic polypeptide and/or insulin. Ultrastructurally, the secretory granules of cells from these tumors were not typical of those found in A-cells from adult human islets. The glucagon-cell tumors from patients without the syndrome were benign, usually multiple, and were small, with diameters from 0.5 mm to 1 cm. In most cases, the cells from these neoplasms arranged in a characteristic pattern (ribbonlike or "gyriform"). In most tumors, the majority of cells showed both glucagon and glicentin immunofluorescence and the ultrastructural appearance of their secretory granules was similar to that of normal islet A-cells. From the morphologic point of view, therefore, cells from tumors not associated with the glucagonoma syndrome resemble normal glucagon cells more closely than those from tumors associated with the syndrome.

Alteration of islet cell populations in spontaneously diabetic mice.

Endocrine-cell populations in the islets of Langerhans of mutant mice with a severe hypoinsulinemic diabetes (ob/ob or db/db on the C57BL/KsJ background) or with a mild hyperinsulinemic diabetes (ob/ob or db/db on the C57BL/6J background) were studied quantitatively by immunofluorescence and morphometry. In severely diabetic mice, islets presented a reduced proportion of insulin containing cells but increased glucagon-, somatostatin-, and pancreatic polypeptide (PP)-containing cells, as compared with islets of control (+/+) mice. An inverse change was observed in islets of mildly diabetic mice: islets were hypertrophic and composed mostly of insulin-containing cells, with decreased proportions of glucagon-, somatostatin-, and PP-containing cells. In both types of diabetic syndromes, the changes in cell populations induced a qualitative alteration of cellular interrelationships in the affected islets.

Calcium and bone homeostasis in heterozygous carriers of CYP24A1 mutations: A cross-sectional study.

BACKGROUND: Bi-allelic CYP24A1 mutations can cause idiopathic infantile hypercalcemia (IIH), adult-onset nephrocalcinosis, and possibly bone metabolism disturbances. It is currently unclear if heterozygous carriers experience clinical problems or biochemical abnormalities. Our objective is to gain insight in the biochemical profile and health problems in CYP24A1 heterozygotes. STUDY DESIGN: Cross-sectional evaluation of participants. Data of previously reported carriers are reviewed. SETTING AND PARTICIPANTS: Outpatient clinic of a tertiary care hospital. Participants were eight family members of an infant with a well-characterized homozygous CYP24A1 mutation c.1186C>T p.(Arg396Trp). OUTCOMES: Serum vitamin D metabolites. Symptoms or biochemical signs of hypercalcemia, hypercalciuria or nephrocalcinosis. Bone health in heterozygous as compared to wild type (WT) subjects. MEASUREMENTS: Genotyping by Sanger sequencing; vitamin D metabolites by liquid chromatography tandem mass spectrometry; renal, calcium and bone markers by biochemical analyses; presence of nephrocalcinosis by renal ultrasound; bone health by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. RESULTS: Six participants were heterozygous carriers of the mutation. None of the heterozygous subjects had experienced IIH. One had a documented history of nephrolithiasis, two others had complaints compatible with this diagnosis. No major differences between WT and heterozygous subjects were found regarding bone health, serum or urinary calcium or 25OHD/24,25(OH)2D ratio. Literature reports on three out of 33 heterozygous cases suffering from IIH. In all three, the 25OHD/24,25(OH)2D ratio was highly elevated. Nephrocalcinosis was frequently reported in family members of IIH cases. LIMITATIONS: Small sample size, lack of a large control group. CONCLUSIONS: Our and literature data suggest that most heterozygous CYP24A1 mutation carriers have a normal 25OHD/24,25(OH)2D ratio, are usually asymptomatic and have a normal skeletal status but may possibly be at increased risk of nephrocalcinosis. A review of the available literature suggests that an elevated 25OHD/24,25(OH)2D ratio may be associated with symptoms of IHH, irrespective of carrier status.

Enzymic and metabolic anomalies in islets of diabetic rats: relationship to B cell mass.

A preferential impairment of the pancreatic B cell secretory response to D-glucose occurs in adult rats injected with streptozotocin during the neonatal period. Three possible explanations for such a preferential defect were investigated in the present study. First, the time course for 3-O-methyl-D-glucose uptake by islets suggested that the anomaly in hexose transport was mainly attributable to a decrease in the space accessible to the D-glucose analog commensurate with the decrease in B cell mass, rather than to a delayed equilibration of hexose concentration across the B cell plasma membrane. Second, the activity of glucose-6-phosphatase was found to be equally low in islets from diabetic and control rats, ruling out the futile cycling between D-glucose and D-glucose 6-phosphate as a cause for the preferential alteration of the secretory response to the hexose. Third, the activity of flavine adenine dinucleotide-linked glycerophosphate dehydrogenase was found to be decreased to a greater relative extent than the B cell mass. This coincided with an impaired generation of 3HOH from L-[2-3H] glycerol in intact islets. It is proposed, therefore, that an altered circulation in the glycerol phosphate shuttle may play a major role in the impaired process of glucose-stimulated insulin release in this model of noninsulin-dependent diabetes.

Human islet cell tumor storing pancreatic polypeptide: a light and electron microscopic study.

A 45-year-old man was operated for surgical treatment of a long-standing peptic ulcer disease and upon inspection of the pancreas for suspected Zollinger-Ellison syndrome, tumor nodules were found in this organ. The tumor tissue examined by immunofluorescence showed specific staining only after incubation with anti-pancreatic polypeptide. Negative results were obtained with antisera directed against insulin, pancreatic glucagon, somatostatin, GLI, VIP, secretin, and gastrin. Examination of the tissue by electron microscopy revealed a homogeneous population of small granule-containing cells. This case, therefore, illustrates a tumor composed of one single hormone-producing cell type and allows definition of the ultrastructural features of human pancreatic polypeptide-containing cells.

Compensatory capabilities of islets of BB/Wor rats exposed to sustained hyperglycemia.

To determine if discordance for autoimmune diabetes in genetically homogeneous animals might reflect differences in the compensatory capacity of their beta cells, the glycemic responses of diabetes-prone BB/Wor rats during a high rate infusion of 50% glucose were compared with normal and with 40% pancreatectomized Wistar rats similarly infused. In all three groups, the initially severe hyperglycemia declined after the first 48 hours to below the target level of 300 mg/dL despite an increasing rate of glucose infusion. The glycemic profile did not differ from controls and was lower than that of the partially depancreatized rats. Five of 20 hyperglycemic BB/Wor rats became diabetic during the 12-day infusion of 50% glucose; there was no difference between their glucose profiles and those of the 15 prediabetic BB/Wor rats that remained nondiabetic throughout the period of hyperglycemic infusion. The latter group of BB/Wor rats, many of which would ultimately have become diabetic, exhibited a 2.4-fold increase in the volume density of their beta cells, compared with a 2.1-fold increase in the Wistar controls. This clinical and morphologic evidence of beta-cell compensation in diabetes-prone rats, even in on the verge of overt diabetes, excludes the possibility that subnormal compensation by beta cells contributes to diabetes in the BB/Wor rat.

Synaptic remodelling in arcuate nucleus after injection of estradiol valerate in adult female rats.

Adult female rats were injected with a single dose (20 mg/kg) of estradiol valerate (EV). The number of synapses was evaluated in thin sections of arcuate nucleus fixed 3, 8, 16 and 32 weeks after EV treatment and compared with the values obtained in the arcuate nucleus of uninjected proestrus control rats. By 8 weeks after EV treatment a significant (P less than 0.001) decrease was found in the number of axo-somatic and axo-dendritic synapses on dendritic shafts, but not in the number of axo-dendritic synapses on dendritic spines. However, by 32 weeks postinjection, the number of axo-somatic and axo-dendritic synapses had returned to control values. This transient decrease in the number of synapses was preceded by a massive appearance of neuronal degenerative images by 3 weeks after EV injection. These results are interpreted as reflecting a process of circuitry remodelling in the arcuate nucleus after a neuronal lesion induced by estrogen.

Freeze-fracture cytochemistry of neuronal membranes: inhomogeneous distribution of filipin-sterol complexes in perikarya, dendrites and axons.

Vibratome slices of cerebellar cortex fixed in a glutaraldehyde solution containing filipin, a sterol-specific probe, were freeze-fractured to study the distribution of filipin-sterol complexes in neuronal plasma membranes. These complexes appear as 25-30 nm protuberances or pits in the fracture face of plasma membranes, and their density was low in dendrites of Purkinje and granule cells. In contrast, the plasma membranes of neuronal perikarya showed an abundant filipin labeling, 4-6 times greater than in dendrites. Parallel fibers, the axons of granule cells also had significantly more filipin-sterol complexes than granule cell dendrites, but fewer than granule cell perikarya. The results reveal difference in the organization of specific regions of neuronal plasma membranes, which are also characterized by a different pattern of synaptic contacts.

Sex differences and maturational changes in arcuate nucleus neuronal plasma membrane organization.

Intramembrane particles (IMP) are believed to represent protein-containing structures in the membrane. Hypothalamic arcuate nucleus neuronal plasma membranes from male and female rats studied from birth to adulthood were quantitatively assessed for IMP number and size using freeze-fracture techniques. We found that newborn female rats have a significantly greater number of IMP than newborn males. There is also a progressive increase in the number of IMP during the first 20 days of postnatal life in both sexes. The rate of protein particle insertion favors females, maintaining the unequal protein particle content into adulthood with female rats having more IMP than males of the same age. The differences in IMP concentration are mainly due to greater numbers of small (less than 10 nm) particles in neuronal membranes from female rats. These data extend previous reports of sexual dimorphism in the arcuate nucleus to the level of plasma membrane organization.

Specific neurons in chick central nervous system stain with an antibody against chick intestinal vitamin D-dependent calcium-binding protein.

A specific antiserum against intestinal vitamin D-dependent calcium-binding protein (CaBP) was used for a systematic immunohistochemical evaluation of immunoreactive sites in the central nervous system of chick. CaBP was observed in the perikarya, dendrites and axons of a specific population of neurons. It is concluded that CaBP represents a marker for selected neurons in the central nervous system of chick.

Neurons with whorl bodies have increased numbers of synapses.

The endoplasmic reticulum in some neurons of the rat hypothalamic arcuate nucleus forms concentric sheets of smooth cisternae which are known as whorl bodies. It has been reported that the number and size of these structures change under varying endocrine conditions. For example, the number of rat arcuate nucleus neurons containing whorl bodies increases after gonadectomy. In studying their relationship to other components of the cell, we found that neuron profiles which contain whorl bodies receive a significantly increased number of axosomatic presynaptic terminals (P less than 0.001). Whorl bodies may mark a subpopulation of endocrine sensitive cells which are characterized by a different pattern of connections and whose response to stimuli includes changes in endoplasmic reticulum organization.

Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system.

A complete mapping of immunoreactive sites for vitamin D-dependent calcium-binding protein (CaBP) was performed on serial sections from the rat central nervous system. CaBP immunoreactivity was found in the perikarya, dendrites and axons of some neurons from the limbic system, from many neurosecretory nuclei, from most sensory nuclei and from the cerebral and cerebellar cortex. In contrast, no CaBP antigenic sites were detectable in the motoneurons of the spinal cord and in those of the cranial nerve nuclei, nor in the neurons from the cerebellar nuclei. A quantitative evaluation revealed a great variability in the number of CaBP-immunoreactive neurons among different areas of the central nervous system. Positive cells represented less than 1% of the neurons in the frontal cortex, whereas 74% of the Purkinje cells from the cerebellar cortex showed immunoreactive staining for CaBP. In addition, 45% of the ependymal cells of the telencephalic ventricles were positive. These data show that CaBP is widely distributed in neurons and ependymal cells from the rat central nervous system although it is more concentrated in some specific areas.

Colchicine injection in the inferior olivary nucleus increases the number of Purkinje cell dendritic spines.

The number of Purkinje cell dendritic spines was evaluated in the cerebellum of rats after injection of colchicine into the inferior olivary nucleus. In these conditions, the spines situated in the proximal (inner) part of the molecular layer were increased as compared to lumicolchicine-injected or non-injected control animals. Most postsynaptic targets for climbing fibers are located in the inner molecular layer and the fact that spines in this region increased when axonal transport was blocked in the climbing fibers suggests that the latter play a role in the control of spine formation.