Stem cells in the development and differentiation of the human adrenal glands

There are no studies on stem cells (SCs) and development and differentiation (DD) of the human adrenal glands. The SCs in DD of the adrenal glands were herein investigated histochemically and immunohistochemically in 18 human embryonic adrenal glands at gestational week (GW) 7–40. At 7 GW, the adrenal glands were present, and at 7 GW, numerous embryonic SCs (ESCs) are seen to create the adrenal cortex. The ESCs were composed exclusively of small cells with hyperchromatic nuclei without nucleoli. The ESCs were positive for neural cell adhesion molecule, KIT, neuron‐specific enolase, platelet‐derived growth factor receptor‐α, synaptophysin, and MET. They were negative for other SC antigens, including chromogranin, ErbB2, and bcl‐2. They were also negative for lineage antigens, including cytokeratin (CK)7, CK8, CK18, and CK19, carcinoembryonic antigen, carbohydrate antigen 19‐9, epithelial membrane antigen, HepPar1, mucin core apoprotein (MUC)1, MUC2, MUC5AC, and MUC6, and cluster differentiation (CD)3, CD45, CD20, CD34, and CD31. The Ki‐67 labeling index (LI) was high (Ki‐67 LI = around 20%). α‐Fetoprotein was positive in the ESCs and adrenal cells. The ESC was first seen in the periphery of the adrenal cortex at 7–10 GW. The ESC migrates into the inner part of the adrenal cortex. Huge islands of ESC were present near the adrenal, and they appeared to provide the ESC of the adrenal. At 16 GW, adrenal medulla appeared, and the adrenal ESCs were present in the periphery or the cortex, in the cortical parenchyma, corticomedullary junctions, and in the medulla. The adrenal essential architecture was established around 20 GW; however, there were still ESCs. At term, there are a few ESCs. These data suggest that the adrenal glands were created by ESCs. Microsc. Res. Tech., 78:59–64, 2015. © 2014 Wiley Periodicals, Inc.     

Immunolocalization of multiple membrane proteins on a carbon replica with STEM and EDX

We present a method for immunolabeling of multiple species of membrane proteins with high spatial resolution. It allows differentiation of equally sized very small markers with different chemical compositions, which leads to high labeling efficiency and reduces steric hindrance of closely spaced immunolabeled biomolecules. Markers such as CdSe/ZnS semiconductor quantum dots and colloidal gold particles are distinguished by differential contrast in high-angle annular detector dark-field STEM mode or by EDX microanalysis of their elemental contents. This method was tested by observation of labeled AMPA- and NMDA-type glutamate receptors on sodium-dodecyl-sulfate-digested replica prepared from rat hippocampus. To improve particle visibility and detectability, the replica films were made exclusively with carbon to avoid the high background of conventional platinum/carbon replica. Extension of the method is suggested by detection of 1.4 nm nanogold particles and its potential application in the biological imaging research.     

Semi-automated imaging system to quantitate estrogen and progesterone receptor immunoreactivity in human breast cancer

A semi‐automated imaging system is described to quantitate estrogen and progesterone receptor immunoreactivity in human breast cancer. The system works for any conventional method of image acquisition using microscopic slides that have been processed for immunohistochemical analysis of the estrogen receptor and progesterone receptor. Estrogen receptor and progesterone receptor immunohistochemical staining produce colorimetric differences in nuclear staining that conventionally have been interpreted manually by pathologists and expressed as percentage of positive tumoral nuclei. The estrogen receptor and progesterone receptor status of human breast cancer represent important prognostic and predictive markers of human breast cancer that dictate therapeutic decisions but their subjective interpretation result in interobserver, intraobserver and fatigue variability. Subjective measurements are traditionally limited to a determination of percentage of tumoral nuclei that show positive immunoreactivity. To address these limitations, imaging algorithms utilizing both colorimetric (RGB) as well as intensity (gray scale) determinations were used to analyze pixels of the acquired image. Image acquisition utilized either scanner or microscope with attached digital or analogue camera capable of producing images with a resolution of 20 pixels /10 μ. Areas of each image were screened and the area of interest richest in tumour cells manually selected for image processing. Images were processed initially by JPG conversion of SVS scanned virtual slides or direct JPG photomicrograph capture. Following image acquisition, images were screened for quality, enhanced and processed. The algorithm‐based values for estrogen receptor and progesterone receptor percentage nuclear positivity both strongly correlated with the subjective measurements (intraclass correlation: 0.77; 95% confidence interval: 0.59, 0.95) yet exhibited no interobserver, intraobserver or fatigue variability. In addition the algorithms provided measurements of nuclear estrogen receptor and progesterone receptor staining intensity (mean, mode and median staining intensity of positive staining nuclei), parameters that subjective review could not assess. Other semi‐automated image analysis systems have been used to measure estrogen receptor and progesterone receptor immunoreactivity but these either have required proprietary hardware or have been based on luminosity differences alone. By contrast our algorithms were independent of proprietary hardware and were based on not just luminosity and colour but also many other imaging features including epithelial pattern recognition and nuclear morphology. These features provide a more accurate, versatile and robust imaging analysis platform that can be fully automated in the near future. Because of all these properties, our semi‐automated imaging system ‘adds value’ as a means of measuring these important nuclear biomarkers of human breast cancer.     

Immunohistochemical characterization of adenosine receptors in rat aorta and tail arteries

Adenosine plays an important role in the cardiovascular system, activating adenosine A(1), A(2A), A(2B), and A(3) receptors, and regulating blood flow either by acting directly on vascular cells or indirectly because of its effects on the central or peripheral nervous systems. The aim of the present study was to investigate whether the pattern of distribution of adenosine receptor subtypes is different on elastic and muscular, using abdominal aorta and tail arteries as models. Immunohistochemistry using anti-A(1), anti-A(2A), anti-A(2B), and anti-A(3) receptor antibodies was performed on perfused-fixed/paraffin-embedded arteries from Wistar rats. 3,3'-Diaminobenzidine tetrahydrochloride (DAB; activated by hydrogen peroxide) staining revealed significant differences in the abundance of A(1), A(2A), and A(3) receptors between abdominal aorta and tail artery and allowed the identification of distinct distribution patterns for A(1), A(2A), A(2B), and A(3) receptors in the tunica adventitia, media, and intima of muscular and elastic arteries. Data are compatible with several previous functional reports supporting that different adenosine receptor subtype expression and/or their distribution in the vessel wall may influence their respective contribution to the control of blood flow.     

Distribution of sex steroid hormone receptors in the avian brain: functional implications for neural sex differences and sexual behaviors

Developmental and seasonal changes in the production of androgens, estrogens, and progestins seem to control sex-specific differentiation and seasonal changes in appetitive and consummatory sexual behaviors of birds. This results in profound sex differences in the quality (sex-specific) or quantity (sex-typical) of behaviors such as courtship, territoriality, or copulation. Steroids affect the brain by binding to intracellularly located receptors. The same brain areas express androgen, estrogen, and progesterone receptors in male and female brains. Sex differences in these genetically determined patterns occur in the size of neuron populations that intrinsically express sex steroid receptors. Further permanent sex differences are subsequent to degenerative fates of receptor expressing neuron populations during ontogeny. Transient sex differences in receptor expression appear to be due to area-specific up- and down-regulation of receptor levels, reflecting transient changes in the level of circulating steroids, changes in environmental conditions, or in the physiological status of the individuals. In particular, intrinsic sex differences in the expression pattern of sex steroid receptors and steroid-independent regulation of the expression level of these receptors in the brain are limiting mechanisms for gonad-dependent sexual development and activities.     

Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibians.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide of the secretin/glucagon/vasoactive intestinal polypeptide superfamily, has been initially characterized in mammals in 1989 and, only 2 years later, its counterpart has been isolated in amphibians. A number of studies conducted in the frog Rana ridibunda have demonstrated that PACAP is widely distributed in the central nervous system (particularly in the hypothalamus and the median eminence) and in peripheral organs including the adrenal gland. The cDNAs encoding the PACAP precursor and 3 types of PACAP receptors have been cloned in amphibians and their distribution has been determined by in situ hybridization histochemistry. Ontogenetic studies have revealed that PACAP is expressed early in the brain of tadpoles, soon after hatching. In the frog Rana ridibunda, PACAP exerts a large array of biological effects in the brain, pituitary, adrenal gland, and ovary, suggesting that, in amphibians as in mammals, PACAP may act as neurotrophic factor, a neurotransmitter and a neurohormone.     

Pharmacological properties of nicotinic acetylcholine receptors in isolated Locusta migratoria neurones

Mechanically dissociated neuronal cell bodies from the thoracic ganglia of Locusta migratoria were viable in culture conditions for up to 2 days and were voltage-clamped to record the effects of GABAergic drugs and physostigmine on the membrane conductance and ACh responses of the dissociated cells. Bicuculline, hydrastine, and gabazine inhibited the EC50 ACh responses of the cells. Both bicuculline and hydrastine were full inhibitors of the ACh responses but gabazine behaved as a partial inhibitor. Bicuculline, hydrastine, and gabazine inhibited the ACh responses in a non-competitive and voltage-independent fashion, suggesting that they are allosteric inhibitors of locust nicotinic ACh receptors. Physostigmine activated currents when applied onto isolated locust neurones. The responses activated by physostigmine were inhibited competitively by tubocurarine, which indicates that physostigmine interacts with the ACh site of locust nicotinic ACh receptors. However, maximal concentrations of physostigmine elicited currents of smaller amplitudes to those evoked by maximal ACh concentrations. Single-channel recordings suggest that the partial efficacy of physostigmine may reflect the low frequency of opening of physostigmine-induced single currents relative to that of ACh-single currents.     

Imaging of immunolabeled membrane receptors in uncoated sem specimens

Epidermal growth factor receptors (EGFR) were labeled with 10 nm immunogold and examined on uncoated specimens of A431 human epidermoid carcinoma cells. A field emission gun and a high-sensitivity YAG ring detector were used to demonstrate the affinity labeling simultaneously in the secondary-electron (SE) and backscattered-electron (BSE) modes with a low accelerating voltage (V_o). At V_o=2kV, the SE and BSE signals were too weak to identify all markers, while at V_o=3–7 kV labeling was observed unambiguously in both the SE and BSE modes with smaller and higher working distances. Increasing the V_o to above 7 kV sometimes provokes instability of the specimens. A V_o of ? 10 kV produces charging artifacts in the SE image, but permits a BSE image of the gold markers providing additional topographic information. In conclusion, immunogold labeling can be used with good results for uncoated specimens.     

Ovarian hormones and fasting differentially regulate pituitary receptors for estrogen and gonadotropin‐releasing hormone in rabbit female

To investigate the mechanisms by which caloric restriction affects reproductive function in female rabbits, we measured, in animals intact or ovariectomized (OVX) estrogen‐primed and fed ad libitum or fasted for 48 h, the adenohypophysial expression of estrogen receptor‐alpha (ESR1) and gonadotropin releasing hormone receptor (GnRHR) and the dynamic secretion of LH following GnRH stimulation. Fasting increased the number of GnRHR‐immunoreactive (‐IR) cells in intact animals, whereas reduced the density of ESR1‐IR cells in OVX rabbits. Estrogen priming decreased the number of ESR1‐IR cells in fasted and OVX animals. Ovariectomy increased the number of ESR1‐IR cells in fed rabbits, but caused an opposite effect in both fed and fasted animals treated with estrogen. Fasting down regulated the mRNA levels for ESR1 and GnRHR. Estrogen‐priming reduced the abundance for ESR1 mRNA in both fed and fasted rabbits, and that for GnRHR in fasted rabbits. Ovariectomy halved ESR1 mRNA levels independently of treatment and feeding condition, whereas increased (P < 001) that for GnRHR in estrogen‐primed rabbits. In all rabbits, an LH surge occurred 30 min after GnRH injection but the lowest levels were found in intact fasted rabbits and the highest in fasted, estrogen‐primed animals. The LH profile was similar in intact and OVX rabbits and neither fasting nor estrogen priming modified it. In conclusion, fasting differentially modifies the ESR1 and GnRHR expression in the pituitary, depending on the presence of gonadal hormones, indicating complex interactions between metabolic signals and ovarian steroids. Microsc. Res. Tech. 77:201–210, 2014. © 2013 Wiley Periodicals, Inc.     

Steroid hormone receptors, matrix metalloproteinases, insulin-like growth factor, and dystroglycans interactions in prostatic diseases in the elderly men

OBJECTIVES: The aim of this study was to evaluate the reactivity of steroid hormone receptors (SHRs), dystroglycans (DGs), matrix metalloproteinases (MMPs), insulin‐like growth factor receptor (IGFR‐1), and laminin (Lam) in both prostatic stromal and epithelial compartments showing different diseases in elderly men. METHODS: Sixty prostatic samples were obtained from 60‐ to 90‐year‐old patients (mean 63 years) with and without prostatic lesions from Hospital of the School of Medicine, State University of Campinas (UNICAMP). The Samples were divided into standard (no lesions); high grade prostatic intraepithelial neoplasia (HGPIN); prostatic cancer (PC); and benign prostatic hyperplasia (BPH) groups. The samples were submitted to immunohistochemistry and Western blotting analyses. Research Ethics Committee of the School of Medicine, University of Campinas/UNICAMP (number 0094.0.146.000‐08). RESULTS: The results showed increased IGFR‐1 and MMPs protein levels in the PC and HGPIN groups. Decreased αDG and βDG protein levels were verified in the PC and HGPIN groups. Androgen receptor (AR) reactivity was similar among all groups. Estrogen receptor α (Erα) immunoreactivity was more intense in the epithelium in the PC and HGPIN groups. Estrogen receptor β (ERβ) immunoreactivity was weak in the epithelium of the HGPIN and PC groups. CONCLUSIONS: To conclude, there was an association among IGFR‐1, MMPs, and SHRs, indicating IGFR‐1 as a target molecule in prostate therapy, considering the IGF proliferative properties. Also, the distinct SHRs reactivities in the lesions in both prostatic compartments indicated different paracrine signals and pointed out the importance of estrogenic pathways in the activation of these disorders. Microsc. Res. Tech. 75:1197–1205, 2012. © 2012 Wiley Periodicals, Inc.     

Receptor binding occurrence and plasma levels of natriuretic peptides in response to sympathectomy

In the present investigation the relationship between the sympathetic nervous system and the endocardial levels of receptor binding sites for natriuretic peptides and the plasma content of atrial natriuretic peptide were analyzed in rats. In order to destruct the cardiac sympathetic nerve terminals, chemical sympathectomy with 6-hydroxydopamine was made in parallel with intravenous measurements of blood pressure and heart frequency. By use of immunohistochemical and enzyme-linked-immunosorbent techniques the expression of tyrosine hydroxylase-positive sympathetic nerve terminals and plasma levels of pro-atrial natriuretic peptide were determined, respectively. The occurrence of receptor binding sites for natriuretic peptides was examined by in vitro receptor autoradiography. In contrast to the marked occurrence of natriuretic peptide receptor binding sites seen in the ventricular endocardium of control rats, the sympathectomized rats exhibited a decreased number of binding sites for natriuretic peptides in the endocardium of both the right and left chambers. Interestingly, this was found in parallel with a significant decrease of systolic and diastolic blood pressure and increased plasma levels of pro-atrial natriuretic peptide in the treated group of rats. These findings, together with those in previous studies, give support to an idea that one part of the blood pressure-decreasing effects, seen in patients treated with beta-adrenergic blockade, might be through a reduction of the natriuretic clearance receptor C, then giving rise to increased levels of atrial natriuretic peptide.     

Physiological significance of gravity receptors on larval cephalic cuticle in the silk moth,Antheraea proylei Jolly

The lateral aspects of larval cephalic cuticle of oak tasar moth, Antheraea proylei, a hybrid between Antheraea pernyi and Antheraea roylei exhibited the presence of gravity receptors in the form of dorsal campaniform sensilla. The distribution pattern and number of dome shaped dorsal campaniform sensilla were found to vary in different larval stages. In the first and second larval stages, 4‐5 sensilla were localized near the apex of the lateral aspects of the cephalic cuticle on either side of the head. From the third larval stage onwards, on the other hand, both the right and left lateral aspects of the cephalic cuticle were covered with innumerable dome shaped dorsal campaniform sensilla. The sensilla were found to be arranged in groups of 3 to 5 on the cephalic cuticle with dome‐free cuticular portion of about 50–100 µm in length between two adjacent groups of the sensilla. The individual dome shaped dorsal campaniform sensillum was either smooth surfaced or was covered with smaller domes through out the surface. The significance of differences in the number and distribution pattern of the dorsal campaniform sensilla among different larval stages in relation to gravity reception and preferred feeding posture are discussed in the light of available literature.     

Immunolocalization of tubulin and actin in thick-sectioned fungal hyphae after freeze-substitution fixation and methacrylate de-embedment

Immunolocalizations in whole mounts of walled cells require an empirically derived, species-specific permeabilization strategy. Particularly when cell walls are recalcitrant to enzymatic digestion, as with many fungi, permeabilization can degrade structure and reduce or destroy antigenicity. To avoid these potential problems, hyphae were freeze-substituted, embedded in a methacrylate resin, thick-sectioned and de-embedded prior to immunolocalization. The cytoskeletal proteins actin and β-tubulin were labelled via indirect immunofluorescence in hyphal tip cells of Magnaporthe grisea , and Trichoderma viride . Well-defined punctate actin plaques were observed concentrated at the subapical cell periphery in both species, especially near the hypha–substratum interface. In addition, a Spitzenkörper core and less intense cytoplasmic binding were observed. Immunoelectron microscopy confirmed localization of actin in the Spitzenkörper core and filasomes. β-Tubulin was immunolocalized as both linear arrays and punctate dots via epifluorescence microscopy. Analysis of 450–550-nm-thick serial sections contributed to improved resolution by the reduction of out-of-focus blur. Overall, the freeze substitution methacrylate de-embedment technique provided superior preservation, serial section analysis, and improved antibody penetration and resolution of these cytoskeletal proteins. The technique will be a valuable tool for use with a variety of reporter molecules in cells of fungi.     

Comparison of transsynaptic viral labeling of central nervous system structures from the uterine horn in virgin, pregnant, and lactating rats

Using the transneuronal viral tracing method, the central nervous system (CNS) connections of the uterine horn were studied in virgin, pregnant, and in lactating rats. The frequency of viral labeling in the brain and the distribution of virus-infected neurons from the uterine horn were compared among groups. There was a marked difference in the frequency of viral labeling in the brain stem. In virgin rats more than half of the brain stems (5 out of 9) were labeled. In contrast, in pregnant animals viral-labeled neurons were detected in only a few cases (3 out of 16) and almost each brain stem of the lactating group was labeled (12 out of 13). A similar, less marked difference was observed in the hypothalamus. The pattern of distribution of infected neurons was similar in each group. In the brain stem, the nucleus of the solitary tract, dorsal motor nucleus of the vagus, area postrema, gigantocellular and paragigantocellular nucleus, ventrolateral medulla, A5 cell group, and caudal raphe nuclei were the most frequently labeled structures. In the diencephalon, viral-infected neurons were detected primarily in the hypothalamic paraventricular nucleus. The telencephalon was devoid of infected cells. Data suggest that the CNS control of the uterine horn varies depending on reproductive status. The low frequency of brain labeling in pregnant rats may be related to the almost complete lack of sympathetic fibers in the uterus prior to parturition and the very high frequency of labeling in lactating animals to the postpartum hyperinnervation of the uterus.     

Immunolocalization of substance P and NK‐1 receptor in hofbauer cells in human normal placenta

Substance P (SP) after binding to the neurokinin‐1 (NK‐1) receptor regulates many biological functions. Both SP and the NK‐1 receptor are expressed in human normal placenta cells, monocytes, and macrophages. However, to our knowledge, the presence of both SP and the NK‐1 receptor in macrophages of the placenta, the Hofbauer cells, is unknown. We demonstrate by immunohistochemistry in human normal placenta samples the presence of both SP and NK‐1 receptors in the cytoplasm and in the nucleus of Hofbauer cells. The findings suggest a functional role of the SP/NK‐1 receptor system in the physiology and pathophysiology of Hofbauer cells in the human placenta. Microsc. Res. Tech. 76:1310‐1313, 2013. © 2013 Wiley Periodicals, Inc.     

Distribution of NGF and NT-3-like protein immunoreactivity in the teleost kidney

By means of immunochemistry and immunohistochemistry, we investigated in the kidney of freshwater and marine teleostean species for the presence and localization of three neurotrophins: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3. In both species studied, NGF-like and NT-3-like immunoreactivity were present in the kidney with different distribution patterns, while BDNF-like immunoreactivity was never detected. In goldfish, NGF-like and NT-3-like immunoreactivity were identified extensively in cells along part of the arterial branches adjacent to the afferent arterioles. In scorpion fish, NGF-like and NT-3-like immunoreactive cells were observed both on afferent arterioles and on adjacent secondary branches derived from renal arteries. No immunoreactivity was detected in other renal structures. A staining pattern of immunoreactivity similar to that obtained for NGF and NT-3 was detected utilizing S100 antibody as a juxtaglomerular (JG) cell marker. Double immunolabellings NGF/S100 and NT-3/S100 evidenced the coexistence of neurotrophin-like proteins and S100-like protein in the same immunoreactive cells, thus identifying them as juxtaglomerular cells. Western blot analysis revealed the presence of molecules immunoreactive to NGF and NT-3, whose molecular weights were very similar to those of the corresponding mammalian neurotrophins. These findings extend the presence and distribution of NGF-like and NT-3-like IR in the kidney to teleost species, suggesting a probable participation of these proteins in the renal functions of freshwater and marine teleosts.     

Distribution of neurotrophin and TrK receptor-like immunoreactivity in the adrenal gland of birds

The occurrence and localization of neurotrophins and their specific TrK receptor-like proteins in the adrenal gland of chicken, duck and ostrich were examined by immunohistochemical methods. In all species studied NGF-, TrK A- and TrK C-like immunoreactivity was observed in neurons and fibers of adrenal ganglia. Thin TrK A- and TrK C-like immunoreactive fibers were also observed among chromaffin cells. NT-3-like immunoreactivity was detected in chromaffin cells as revealed by the double immunolabelings NT-3/chromogranin A and NT-3/DbetaH. The interrenal tissue never showed IR to any neurotrophins and TrK tested, and none of the adrenal structures displayed immunoreactivity to BDNF and TrK B. Double immunolabelings NGF/TrK A, NGF/TrK C and TrK A/TrK C showed colocalization in some neurons and fibers in adrenal ganglia. In adrenal glands of the species studied, the distribution of neurotrophins and TrK receptors could suggest an involvement of NT-3 on neuronal populations innervating adrenal ganglia by means of its high affinity receptor TrK C and low affinity receptor TrK A. In addition, NGF could be utilized by neuronal populations of adrenal ganglia through its preferential receptor TrK A by an autocrine or paracrine modality of action.