Increased density of inhibitory noradrenergic parenchymal nerve fibers in hypertrophic islets of Langerhans of obese mice.

BACKGROUND AND AIM: We sought to identify mechanisms of beta cell failure in genetically obese mice. Little is known about the role of pancreatic innervation in the progression of beta cell failure. In this work we studied adrenergic innervation, in view of its potent inhibitory effect on insulin secretion. We analyzed genetically obese ob/ob and db/db mice at different ages (6- and 15-week-old), corresponding to different compensatory stages in the course of beta cell dysfunction. 15 week-old HFD mice were also studied. METHODS AND RESULTS: All mice were characterized by measures of plasma glucose, insulin, and HOMA. After perfusion, pancreata were dissected and studied by light microscopy, electron microscopy, and morphometry. Insulin, Tyrosine Hydroxylase-positive fibers and cells and Neuropeptide Y-positive cells were scored by immunohistochemistry. Islets of obese mice showed increased noradrenergic fiber innervation, with significant increases of synaptoid structures contacting beta cells compared to controls. Noradrenergic innervation of the endocrine area in obese db/db mice tended to increase with age, as diabetes progressed. In ob/ob mice, we also detected an age-dependent trend toward increased noradrenergic innervation that, unlike in db/db mice, was unrelated to glucose levels. We also observed a progressive increase in Neuropeptide Y-immunoreactive elements localized to the islet core. CONCLUSIONS: Our data show increased numbers of sympathetic nerve fibers with a potential to convey inhibitory signals on insulin secretion in pancreatic islets of genetically obese animals, regardless of their diabetic state. The findings suggest an alternative interpretation of the pathogenesis of beta cell failure, as well as novel strategies to reverse abnormalities in insulin secretion.

The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.

The origin of brown adipocytes arising in white adipose tissue (WAT) after cold acclimatization is unclear. Here, we demonstrate that several UCP1-immunoreactive brown adipocytes occurring in WAT after cold acclimatization have a mixed morphology (paucilocular adipocytes). These cells also had a mixed mitochondrioma with classic "brown" and "white" mitochondria, suggesting intermediate steps in the process of direct transformation of white into brown adipocytes (transdifferentiation). Quantitative electron microscopy disclosed that cold exposure (6 degrees C for 10 days) did not induce an increase in WAT preadipocytes. beta(3)-adrenoceptor-knockout mice had a blunted brown adipocyte occurrence upon cold acclimatization. Administration of the beta(3)-adrenoceptor agonist CL316,243 induced the occurrence of brown adipocytes, with the typical morphological features found after cold acclimatization. In contrast, administration of the beta(1)-adrenoceptor agonist xamoterol increased only the number of preadipocytes. These findings indicate that transdifferentiation depends on beta(3)-adrenoceptor activation, whereas preadipocyte recruitment is mediated by beta(1)-adrenoceptor. RT-qPCR experiments disclosed that cold exposure induced enhanced expression of the thermogenic genes and of genes expressed selectively in brown adipose tissue (iBAT) and in both interscapular BAT and WAT. beta(3)-adrenoceptor suppression blunted their expression only in WAT. Furthermore, cold acclimatization induced an increased WAT expression of the gene coding for C/EBPalpha (an antimitotic protein), whereas Ccna1 expression (related to cell proliferation) was unchanged. Overall, our data strongly suggest that the cold-induced emergence of brown adipocytes in WAT predominantly reflects beta(3)-adrenoceptor-mediated transdifferentiation.

Noradrenergic parenchymal nerve fiber branching after cold acclimatisation correlates with brown adipocyte density in mouse adipose organ.

The mammalian adipose organ is composed of subcutaneous and visceral depots containing white and brown adipocytes. Cold acclimatisation induces an increase in the brown component without affecting the overall number of adipocytes; this form of plasticity is associated to obesity and diabetes resistance in experimental models. Cold activates the drive of the sympathetic nervous system to the adipose organ, where the vast majority of nerve fibers are in fact noradrenergic. However, it is unclear whether and how such fibers are involved in the plastic changes of the adipose organ. We thus conducted a systematic study of the distribution and number of sympathetic noradrenergic nerve fibers in the adipose organ of mice kept at different environmental temperatures. Adult Sv129 female mice were kept at 28 degrees C or 6 degrees C for 10 days. The density of tyrosine hydroxylase (noradrenergic)-positive nerve fibers (no. of fibers per 100 adipocytes) was calculated in the subcutaneous and visceral depots of the adipose organ, and a correlation was sought between fiber density and proportion of brown adipocytes. Tyrosine hydroxylase-positive parenchymal fibers were detected in all subcutaneous and visceral depots among white as well as brown adipocytes, the mediastinal depot displaying the densest innervation. Cold acclimatisation induced a threefold increase in the total number of TH fibers in the whole organ. The proportion of brown adipocytes positively correlated with noradrenergic fiber density in the organ. Taken together, these data suggest that cold acclimatisation induces noradrenergic fiber branching in the adipose organ of adult mice, and that such changes may be a precondition for its plastic transformation into a brown phenotype.

Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice.

Accumulation of visceral fat is a key phenomenon in the onset of obesity-associated metabolic disorders. Macrophage infiltration induces chronic mild inflammation widely considered as a causative factor for insulin resistance and eventually diabetes. We previously showed that >90% of macrophages infiltrating the adipose tissue of obese animals and humans are arranged around dead adipocytes, forming characteristic crown-like structures (CLS). In this study we quantified CLS in visceral and subcutaneous depots from two strains of genetically obese mice, db/db and ob/ob. In both strains, CLS were prevalent in visceral compared with subcutaneous fat. Adipocyte size and CLS density exhibited a positive correlation both in visceral and in subcutaneous depots; however, the finding that adipocyte size was smallest and CLS density highest in visceral fat suggests a different susceptibility of visceral and subcutaneous adipocytes to death. Visceral fat CLS density was 3.4-fold greater in db/db than in ob/ob animals, which at the age at which our experimental strain was used are more prone to glucose metabolic disorders.

Stages of development of the ribosome-lamella complex: an ultrastructural study.

Ribosome-lamella complexes (RLC) are intracytoplasmic organelles observed in a wide variety of disorders, but mostly in hematologic malignancies. Although their close topographic relationship with rough endoplasmic reticulum (RER) suggests their derivation from it, their development and functional role are unclear. Their maturation phases were studied in 20 cases (19 hematologic neoplasms and 1 parathyroid adenoma) where electron microscopy had evidenced their presence. In 19 of thesecases, RLC were in an advanced stage of maturation, whereas in one (acute monoblastic leukemia) they were observed in the early stages of development and appeared to arise from peculiar RER configurations within blast cells, which were rich in both organelles. In this case, the authors observed numerous RER cisternae with distinctive cylindric, concentric and/or whorl configurations, RLC associated and not associated with these configurations, and intermediate structures. The latter were characterized by lamellae devoid of ribosomes oriented parallel to the RER configurations. Reticulum configurations were observed in no other case. The ultrastructural aspects observed in these 20 cases suggest that RLC synthesis proceeds as follows: (1) arrangement of RER in cylindric configurations; (2) synthesis of lamellae oriented parallel to the cylindric configurations (pre-RLC); (3) formation of RLC when ribosomes appear between the lamellae associated with configurations (immature RLC); (4) formation of mature RLC with disappearance of the reticulum.

Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes.

Multilocular, mitochondria-rich adipocytes appear in white adipose tissue (WAT) of rats treated with the beta3-adrenoceptor agonist, CL-316243 (CL). Objectives were to determine whether these multilocular adipocytes derived from cells that already existed in the WAT or from proliferation of precursor cells and whether new mitochondria contained in them were typical brown adipocyte mitochondria. Use of 5-bromodeoxyuridine to identify cells that had undergone mitosis during the CL treatment showed that most multilocular cells derived from cells already present in the WAT. Morphological techniques showed that at least a subpopulation of unilocular adipocytes underwent conversion to multilocular mitochondria-rich adipocytes. A small proportion of multilocular adipocytes ( approximately 8%) was positive for UCP1 by immunohistochemistry. Biochemical techniques showed that mitochondrial protein recovered from WAT increased 10-fold and protein isolated from brown adipose tissue (BAT) doubled in CL-treated rats. Stained gels showed a different protein composition of new mitochondria isolated from WAT from that of mitochondria isolated from BAT. Western blotting showed new mitochondria in WAT to contain both UCP1, but at a much lower concentration than in BAT mitochondria, and UCP3, at a higher concentration than that in BAT mitochondria. We hypothesize that multilocular adipocytes present at 7 days of CL treatment have two origins. First, most come from convertible unilocular adipocytes that become multilocular and make many mitochondria that contain UCP3. Second, some come from a cell that gives rise to more typical brown adipocytes that express UCP1.

Effects of long-term treatment with human pure follicle-stimulating hormone on semen parameters and sperm-cell ultrastructure in idiopathic oligoteratoasthenozoospermia.

Ten subfertile men affected by idiopathic oligoteratoasthenozoospermia and exhibiting normal serum hormone levels received a long-term treatment with human pure follicle-stimulating hormone (hp-FSH) (150 IU, intramuscularly, three times per week for 6 months). Semen parameters and ultrastructural features of spermatozoa were evaluated before and after therapy. The results showed an increase in sperm cell concentration and, more interestingly, motility. Electron microscopic examination revealed an improved fine architectural pattern, mainly involving acrosome, head and chromatin and middle-piece, in accordance with the positive changes of functional data. No significant changes of hp-FSH treatment on serum hormone levels were observed, since the latter were found to be substantially unchanged after 6 months of therapy. The present data suggest: (i) the benefit of hp-FSH administration in idiopathic oligoteratoasthenozoospermia, when hormone parameters support a substantial integrity of spermatogenetic microenvironment and (ii) an optimal effect after long-term (6 months) therapy.

The retractile testis can be a cause of adult infertility.

OBJECTIVE: To determine the functional state of the testes of young adults treated for small and hypotrophic retractile testis at prepubertal age by orchiopexy and/or hormonal therapy and the functional state of the testes of adults with retractile testis. DESIGN: Spermiogram and transmission electron microscopy (TEM) study of the semen. Light microscopic and TEM studies of semen and testicular biopsies of adult with retractile testis were performed. SETTING: Division of Pediatric Surgery in an academic environment. PATIENT(S): Thirty-eight young adults (mean age, 18 years) treated for retractile testis at prepubertal age and seven adults (mean age, 28 years) with retractile testis. INTERVENTION(S): Two cycles of hCG, followed by surgical therapy (orchiopexy) when unsuccessful. MAIN OUTCOME MEASURE(S): Fertility of young adults treated for retractile testis at prepubertal age. RESULT(S): Only 8 of 38 (21%) young adults had normal spermiograms. Five of 38 (13%) were azoospermic and 25 of 38 (66%) were oligoasthenozoospermic with ultrastructural signs of altered maturation of the sperm and a higher number of atypical forms. Of the adults with retractile testis, 2 of 7 (28.5%) were normal, 3 of 7 (43%) were oligoasthenozoospermic, and 2 of 7 (28%) were azoospermic. CONCLUSION(S): Our data support the hypothesis that prepubertal retractile testis showing signs of reduced consistency and size is a risk factor for adult infertility and requires treatment.

Cryptorchidism and infertility: retrospective study of 18 post puberal patients affected with monolateral cryptorchidism.

The Authors report their experience on patients with monolateral inguinal cryptorchidism, comparing the clinical picture with the morphological pattern of both the testicular parenchima and the semen. Light and electron microscopic analysis of testicular biopsy and spermatozoa from the ejaculate have been taken into account. 4 patients out of 18 (22.2%) showed a normal spermiogram, while 14 showed a pathological spermiogram. The morphological study of the testicular parenchima showed a spectrum of lesions, most of which advanced, confirming that they are irreversible lesions of the germinal epithelium.

Subcutaneous nodules in a patient hyposensitized with aluminium-containing allergen extracts: a microanalytical study.

Electron spectroscopic imaging (ESI) is a recent tool for electron microscopic analysis which permits the recognition of the distribution of elements in a specimen, with a spatial resolution up to 5 nm. The authors performed the ESI together with X-ray microanalysis (EDX) and transmission electron microscopy to study granulomatous subcutaneous nodules in a patient who had undergone a desensitising therapy with an aluminium-containing vaccine. Aluminium was detected both by EDX and ESI, mainly in lysosomes of histiocytes containing needle-shaped material in a lucent matrix. However, ESI provided a better localization of aluminium with respect to EDX. This result suggests that the identification of aluminium-containing structures cannot be obtained only by ultrastructural morphology and underlines the utility of a microanalitycal study for a correct diagnosis in the presence of a needle-shaped deposition of dense material in lysosomes.

Immunohistochemical, ultrastructural and morphometric evidence for brown adipose tissue recruitment due to cold acclimation in old rats.

OBJECTIVE: The aim of this work was to study the reactivity to chronic cold stress of interscapular brown adipose tissue (IBAT) in old rats by stereological, immunohistochemical and ultrastructural methods. DESIGN: Five 2-year-old rats were cold-acclimated at 4 degrees C for 4 weeks and six rats were used as controls (20-23 degrees C). MEASUREMENTS: The following were measured: IBAT volume and weight; unilocular and multilocular brown adipocyte content; preadipocyte number; multilocular cell mitochondrial area; cristae length and density per mitochondrion, and immunoreactivity for the brown adipose tissue specific uncoupling protein (UCP). RESULTS: Following cold acclimation, IBAT increased significantly in weight, volume, relative mass and number of multilocular adipocytes (170%). The number of unilocular adipocytes did not vary significantly. Multilocular adipocytes of both cold-acclimated rats and controls expressed the uncoupling protein, but in the experimental group cristae length and density per mitochondrion were significantly higher. Multilocular adipocyte precursors were observed in only one cold-acclimated rat but not in controls. CONCLUSION: The response of brown adipose tissue of old rats to chronic cold stimulus is similar to that observed in young and adult rats. Cold acclimation induces brown adipocyte recruitment: their number increases significantly, they test UCP-positive and their mitochondria are significantly more active than in controls. On the other hand, the number of unilocular adipocytes is not significantly affected, which may serve to improve the utilization of the heat produced by thermogenesis.

Quantitative evaluations of gap junctions in old rat brown adipose tissue after cold acclimation: a freeze-fracture and ultra-structural study.

The morphological and functional modifications of brown adipose tissue (BAT), the tissue responsible for non-shivering thermogenesis, are well established during the phases of active stimulation (i.e. neonatal period and cold acclimation) in young animals. The 'active' brown adipocytes are filled with numerous small lipid vacuoles and large mitochondria packed with cristae rich in the protonophore uncoupling protein (UCP), whereas the 'quiescent' cell shows larger, confluent vacuoles and smaller mitochondria with rarefied cristae poor of the uncoupling protein. It is well known from literature that also gap junctions (gjs), responsible for the electrical coupling among adjacent adipocytes, modify their size following the physiological stimulus in young animals. This is in agreement with the morphology of the functionally active brown adipocyte, i.e. the multilocular, UCP-positive cell. Although the presence of the BAT in old animals is well documented, less is known about its reactivity to physiological stimuli. The present work demonstrates that after cold acclimation brown adipocytes of old rats (2 years) change their ultrastructure in a similar way as in young rats. A quantitative analysis of gap junction areas on replicas obtained by the freeze fracture technique, showed that gj increase in size (mean area 53.2 vs 110.4 x 10(-3) microns2, p = 0.003). All these morphological modifications are quite similar to those observed in BAT of young and young adult rats, supporting the hypothesis of a physiological role of brown adipose tissue at every age.

S-100 protein in rat brown adipose tissue under different functional conditions: a morphological, immunocytochemical, and immunochemical study.

The brown adipose tissue (BAT) is responsible for nonshivering thermogenesis, exhibiting changes in cell morphology related to the functional conditions. In stimulated BAT (neonatal period, cold acclimation) the large majority of adipocytes become multilocular and active, while in inactive BAT (warm acclimation) most cells are pauci- or unilocular and inactive, very similar to white adipocytes. It is well known that white adipocytes are sites of concentration of S-100, a calcium-binding protein originally isolated from the nervous system and later detected also in nonneural cell types, whereas data on the possible presence of this protein in BAT are lacking. The present study used morphological, immunocytochemical, and immunochemical methods to investigate the presence of S-100 protein in BAT under different functional conditions. We found that S-100 was present in both stimulated and inactive BAT and that is was expressed in significantly higher quantities in the latter than in the former. The multilocular cells were always negative (in both active and inactive tissue), whereas the pauci- and unilocular cells were always S-100-positive under both functional conditions. These data suggest that only pauci- and unilocular brown adipocytes express S-100 protein, thus manifesting a possible relationship between S-100 and cell morphology.

The normal, cryptorchid and retractile prepuberal human testis: a comparative morphometric ultrastructural study of 101 cases.

Fifty-two surgical biopsies from retractile testes of patients in pediatric age (3-14 years), of which 25 were treated with hormonal therapy (RT) and 27 did not undergo therapy before orchidopexy (RNT), were compared with the biopsies of 18 normal (N) and 30 cryptorchid or ectopic (E) testes. A light and electron microscopic morphologic and morphometric study was performed. For the quantitative investigation 4 parameters were selected: a) the mean tubular diameter (on 20 cross-sections); b) the mean spermatogonial number per tubular section; c) the mean nucleolar area of the Sertoli cells; and d) the mean thickness of the tubular basal lamina. The 101 biopsies were collected for statistical evaluation into four age groups: 3-6 years, 7-10, 11-13 without spermatogenesis and 10-14 with signs of early spermatogenesis. In the RT category the mean tubular diameter and the mean spermatogonial number were similar to N in the first two age groups, but were significantly reduced in the RNT categories. The morphometric study of the Sertoli cell nucleolar area confirms the delay of maturation observed in the categories of RT, RNT and E. In normal biopsies, the basal lamina shows a progressive reduction of the thickness, with the lowest values around puberty, while constantly higher values were found in the other categories, although this increase is not statistically significant.

Ultrastructure of pituitary folliculo-stellate cells of lactating rat during treatment with 2-bromo-alpha-ergocriptine.

Folliculo-stellate cells of adenohypophysis were studied by transmission electron microscopy in lactating rats during treatment with the dopaminergic agonist 2-bromo-alpha-ergocriptine. Folliculo-stellate cells showed marked activation in control lactating rats, were unaffected by a single injection of the drug (3 mg/kg), regressed to the morphology of those found in non-lactating animals upon 6 days of drug treatment (1 mg/kg/die) both in presence and in absence of the suckling stimulus. However, morphometric analysis showed a proportion of folliculo-stellate cells in the gland which was similar in the four experimental groups (range: 11.3-13.5%), despite the increasing reduction of prolactin cells (55, 44, 32, 30%). In chronically treated animals to which suckling had been denied, extruding and degenerating prolactin cells were found, as well as extracellular material in non follicular spaces; the latter were lined by both folliculo-stellate and endocrine cells. These data suggest a dual reactivity of folliculo-stellate cells in the adenohypophysis according to the functional status of the gland.

Reproducibility of mitotic counts and identification of mitotic figures in malignant glial tumors.

Quantitative pathology is a rapidly growing field of pathology. This field is developing methods allowing greater objectivity and better reproducibility in diagnostic decisions. The introduction of quantitative pathology as a teaching subject in medical education will help this field to become more widely and more correctly applied. Courses on quantitative pathology are organized yearly by the Department of Pathology at the University of Ancona. The 1986 course emphasized training of students in understanding basic problems such as variation caused by tissue processing, instrumentation, data handling and interpretation, and variation between observers. In this paper we describe experiments performed during the course on identification and quantitation of mitoses in malignant glial tumors. The participants, i.e. students of the postgraduate Pathology School, could identify mitoses with a 'substantial' reproducibility. However, when the observers were allowed freely to choose the fields to count, the reproducibility was 'slight'. The study suggests that sampling rules should be applied which secure reproducible application of morphometric method also on condition of free selection of fields. Because the application of such rules needs expertise in histopathology, it seems that morphometric method can best be applied by histopathologists themselves.

An immunohistochemical study in thyroid cancer.

Follicular, papillary, anaplastic and medullary cancers of the thyroid were investigated using immunohistochemical methods. The following antibodies were used: anti-S-100, antineuron-specific enolase (NSE), antikeratin, antithyroglobulin, anticalcitonin, anticarcinoembryonic antigen (CEA), antiepithelial membrane antigen (EMA); the following hormones were also tested in the medullary carcinoma: gastrin, ACTH and serotonin. Papillary and follicular carcinoma in particular reacted with anti-S-100 and anti-NSE; the anaplastic neoplasia reacted with anti-S-100 (25%), anti-NSE (12%), antikeratin (12%), antithyroglobulin (12%), anti-CEA (37%) and anti-EMA (37%). Medullary carcinoma reacted with anticalcitonin (100%), anti-CEA (96%), anti-NSE (79%), anti-EMA (4%) and anti-S-100 (17%). We were not able to correlate the virulence of the medullary carcinoma with the anticalcitonin and anti-CEA reactivity, while the hyperplastic C cells were immunoreactive both with calcitonin or with CEA.

Benign and malignant mesothelial lesions of the pleura: quantitative study.

Benign and malignant mesothelial lesions of the pleura are quantitatively studied using a Leitz TAS image analyzing system. The Feulgen fast green staining method allows performing on the same cell measurements of the shape and size of the nucleus, the size of the nucleolus and the content of DNA. Numerical data are studied in a multivariate statistical analysis in order to obtain the probability of the morphometric diagnosis in each case. Results demonstrate that the most important measurements are the percentage of cells with evident nucleolus, the nuclear perimeter and the standard deviation of the DNA content, and that these measurements give a correct diagnosis in all cases.