Light Microscopic Immunocytochemistry Research Articles

Β-endorphin-immunoreactive perikarya appear to receive innervation from NPY-immunoreactive fiber varicosities in the human hypothalamus.

Morphological and pharmacological studies indicate that hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons communicate with each other in rats and regulate a variety of hypothalamic and extrahypothalamic functions. Indeed, electron microscopic studies revealed NPY-immunoreactive (NPI-IR) synapses on β-endorphin-IR neurons in the hypothalamus. However, no such connections have been reported in humans. Here, we studied the putative NPY-β-endorphin associations with high-resolution light microscopic double-label immunocytochemistry in the human hypothalamus. The majority of β-endorphin-IR perikarya appear to be innervated by abutting NPY-IR fibers in the infundibulum/median eminence, receiving more than 6 contacts (38% of the counted neurons) or three to six contacts (42% of the counted neurons). The rest of the β-endorphin-IR neurons are lightly innervated by NPY fibers (14%, one-three contacts) or do not receive any detectable NPY-IR axon varicosities (6% of the counted neurons). Since β-endorphin is cleaved from the proopiomelanocortin (POMC) precursor, the NPY-β-endorphin connections also provide the foundation for NPY-α-MSH and NPY-ACTH connections and their subsequent physiology. The close anatomical connections between NPY-IR nerve terminals and β-endorphin-IR neurons reported herein may represent functional synapses and provide the foundation for NPY-stimulated β-endorphin release. By interacting with β-endorphin, NPY may have a more widespread regulatory capacity than acting alone on different neurotransmitter systems.

Castration causes an increase in lysosomal size and upregulation of cathepsin D expression in principal cells along with increased secretion of procathepsin D and prosaposin oligomers in adult rat epididymis

In the epididymis, lysosomal proteins of the epithelial cells are normally targeted from the Golgi apparatus to lysosomes for degradation, although their secretion into the epididymal lumen has been documented and associated with sperm maturation. In this study, cathepsin D (CatD) and prosaposin (PSAP) were examined in adult epididymis of control, and 2-day castrated rats without (Ct) and with testosterone replacement (Ct+T) to evaluate their expression and regulation within epididymal epithelial cells. By light microscope-immunocytochemistry, a quantitative increase in size of lysosomes in principal cells of Ct animals was noted from the distal initial segment to the proximal cauda. Androgen replacement did not restore the size of lysosomes to control levels. Western blot analysis revealed a significant increase in CatD expression in the epididymis of Ct animals, which suggested an upregulation of its expression in principal cells; androgens restored levels of CatD to that of controls. In contrast, PSAP expression in Ct animals was not altered from controls. Additionally, an increase in procathepsin D levels was noted from samples of the epididymal fluid of Ct compared to control animals, accompanied by an increased complex formation with PSAP. Moreover, an increased oligomerization of prosaposin was observed in the epididymal lumen of Ct rats, with changes reverted to controls in Ct+T animals. Taken together these data suggest castration causes an increased uptake of substrates that are acted upon by CatD in lysosomes of principal cells and in the lumen by procathepsin D. These substrates may be derived from apoptotic cells noted in the lumen of proximal regions and possibly by degenerating sperm in distal regions of the epididymis of Ct animals. Exploring the mechanisms by which lysosomal enzymes are synthesized and secreted by the epididymis may help resolve some of the issues originating from epididymal dysfunctions with relevance to sperm maturation.

The Cytoplasmic Droplets of Rat Epididymal Spermatozoa Show Diminution of Glycosyl Transferases During Spermatozoa Epididymal Maturation


 
 
 
 The cytoplasmic droplet of the epididymal spermatozoon is a small localized outpouching of cytoplasm of the tail and is of unknown significance. Electron microscopy revealed flattened saccular elements of almost exclusive membranous components. In this study, a simple, reproducible method for the isolation of saccular elements from the caput and the cauda of rat epididymal spermatozoa is presented. Using exogenous glycosylation assays and immunocytochemistry for light and electron microscopy, we confirm the presence of three Golgi markers in the saccular elements of rat cytoplasmic droplets, namely, a-2,6-sialyltransferase, b- 1,4-galactosyltransferase and TGN-38 (trans-Golgi network protein). The variation in galactosyltransferase activity of isolated saccular elements in response to in vitro trypsinization was similar to that observed in the rat liver Golgi apparatus. Electrophoresis and immunoblotting analyses revealed polypeptide modifications in the saccular elements during spermatozoa epididymal transit. In addition, a significant decline in the content of galactosyltransferase and sialyltransferase occurred in the saccular elements during spermatozoa transit from the caput to the cauda epididymides. The content of TGN-38, however, did not show any significant change, suggesting that the modification of the content of the saccular elements is selective. Considering the glycosylating ability of the saccular elements and that the diminution of glycosyltransferase activities in cytoplasmic droplets coincides with the maturation of spermatozoa during epididymal transit, we suggest a potential role for the saccular elements of cytoplasmic droplets in the maturation of spermatozoa during their transit through the epididymis.
 
 
 

Thyrotropin-releasing hormone axonal varicosities appear to innervate dopaminergic neurons in the human hypothalamus.

Thyrotropin-releasing hormone (TRH) has a critical role in the central regulation of thyroid-stimulating hormone (TSH) from the anterior pituitary, and subsequently, thyroid hormone secretion from the thyroid gland. In addition to its role in the regulation of HPT axis, TRH is a potent regulator of prolactin (PRL) secretion by stimulating PRL secretion either directly from lactotrophs or indirectly via its action on the tuberoinfundibular dopamine (TIDA) neurons. In rodents, the TRH neurons which regulate TSH and thyroid hormone secretion, called hypophysiotropic TRH neurons, are in the medial subdivision of the parvicellular paraventricular nucleus (PVN). In humans, the PVN also contains a large population of TRH neurons, especially in its medial part, but the location of hypophysiotropic TRH neurons is not yet known. In addition to regulating TSH and PRL secretion, TRH also functions as a neurotransmitter/neuromodulator. In rodents and teleosts, TRH axons densely innervate TIDA neurons to inhibit tyrosine hydroxylase (TH) biosynthesis, neuronal firing, and dopamine turnover which may contribute to increasing PRL secretion. No such connections have been reported in humans, although dopaminergic neurons express TRH receptors and TRH also regulates PRL secretion. The objectives of this study were to map TRH-IR and TH-IR structures in the human hypothalamus with single-label light microscopic immunocytochemistry and study their interaction with double-label light microscopic immunocytochemistry. We show that TRH-IR nerve terminals densely surround TH-IR neurons (perikarya and dendrites) in the infundibulum of the human hypothalamus. The micrographs illustrating these juxtapositions were taken by Olympus BX45 microscope equipped with a digital camera and with 100X oil immersion objective. Composite images were created from the consecutive micrographs if the neurons were larger than the frame of the camera, using Adobe Photoshop software. As no gaps between TRH-IR and TH-IR elements were seen, these contacts may be functional synapses by which TRH regulates the activity of dopaminergic neurons and subsequently TSH and PRL secretion.

Localization of NUCB2/Nesfatin‐3/Nesfatin‐1 in Normal and Inflamed Human and Mouse Lungs, and Human Neutrophils

Nesfatin‐1 regulates hunger and fat storage and is produced in the hypothalamus of mammals. Nucleobindin2 (NUCB2)/nesfatin‐1 protein expression in human plasma positively correlates with expression of pro‐inflammatory cytokines in patients with chronic obstructive pulmonary disease (COPD) suggesting its potential role in lung inflammation. However, we still don’t precisely know the expression of NUCB2/nesfatin‐1 in lungs and neutrophils. In this study, the expression of NUCB2/nesfatin‐3/nesfatin‐1 in normal and inflamed human and mouse lungs and neutrophils were examined with light and electron microscopic immunocytochemistry. Results from light microscopy showed that the localization of NUCB2/nesfatin‐1 in the pulmonary epithelium, alveolar septa, vascular endothelium and various immune cells does not change with inflammatory status. Electron microscopy revealed constitutive localization within the nucleus and cytoplasm of the aforementioned cells. Further, NUCB2/nesfatin‐1 accumulated within 0.5μm of the plasma membrane in human neutrophils following 90mins of 1ng/mL LPS stimulation. NUCB2/nesfatin‐3 was also found to localize in euchromatic portions of neutrophilic nuclei at 5 times the mean concentration compared to heterochromatin. Finally, our results indicate that NUCB2/nesfatin‐3 is predominantly cytoplasmic as it localizes at 2 times the concentration in neutrophilic cytoplasm compared to nucleus. Our study is the first to detail the localization of NUCB2/nesfatin‐1/nesfatin‐3 in lungs and neutrophils, and also implicate their potential role in transcriptional regulation.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada

Immunogold labelling reveals intense distribution of hyaluronate in the regenerating fin blastema of the goldfish

AbstractImmunogold labelling reveals intense distribution of hyaluronate in the regenerating fin blastema of the goldfish (Acta Zoologica, Stockolm). Caudal fin regeneration in goldfish has been analysed at 3–16 days after tail amputation by light and electron microscopy immunocytochemistry. Hyaluronate is detected using a biotinilated hyaluronate binding protein and anti‐biotin antibodies coupled to gold and silver intensification. Hyaluronate appears among migrating keratinocytes of the wound epidermis and mesenchymal cells of the blastema at 3–6 days post‐amputation. Immunofluorescence for 5BrdU indicates that most proliferation of the wound epidermis and blastema occurs among mesenchymal and bone cells of the amputated lepidotrichia. Few keratinocytes proliferate indicating they mainly derive by migration from the stump borders. Among blastema and epithelial cells, numerous apparently pycnotic cells are present at 3–6 days of regeneration, most identified as immune cells. Silver intensification clearly shows that blastema cells are surrounded by hyaluronate suggesting that this glycosaminoglycan limits the contact between immune and blastema cells, perhaps avoiding an immune reaction that would damage survival of the blastema. Hyaluronate labelling decreases between connective and bone cells in proximal regions at 14–16 days post‐amputation and is low to absent in normal stump tissues. The study suggests that a high level of hyaluronate located among cells is essential for the formation of a regenerating blastema in fish and in other vertebrates.

Dopamine Regulation of GABAA Receptors Contributes to Light/Dark Modulation of the ON-Cone Bipolar Cell Receptive Field Surround in the Retina

Cone bipolar cells are interneurons that receive synaptic input from cone photoreceptor cells and provide the output of the first synaptic layer of the retina. These cells exhibit center-surround receptive fields, a prototype of lateral inhibition between neighboring sensory cells in which stimulation of the receptive field center excites the cell whereas stimulation of the surrounding region laterally inhibits the cell. This fundamental sensory coding mechanism facilitates spatial discrimination and detection of stimulus edges. However, although it is well established that the receptive field surround is strongest when ambient or background illumination is most intense, e.g., at midday, and that the surround is minimal following maintained darkness, the synaptic mechanisms that produce and modulate the surround have not been resolved. Using electrical recording of bipolar cells under experimental conditions in which the cells exhibited surround light responses, and light and electron microscopic immunocytochemistry, we show in the rabbit retina that bright-light-induced activation of dopamine D1 receptors located on ON-center cone bipolar cell dendrites increases the expression and activity of GABAA receptors on the dendrites of the cells and that surround light responses depend on endogenous GABAA receptor activation. We also show that maintained darkness and D1 receptor blockade following maintained illumination and D1 receptor activation result in minimal GABAA receptor expression and activity and greatly diminished surrounds. Modulation of the D1/GABAA receptor signaling pathway of ON-cBC dendrites by the ambient light level facilitates detection of spatial details on bright days and large dim objects on moonless nights.

Subcellular organization of UBE3A in neurons

ABSTRACTUbiquitination regulates a broad array of cellular processes, and defective ubiquitination is implicated in several neurological disorders. Loss of the E3 ubiquitin–protein ligase UBE3A causes Angelman syndrome. Despite its clinical importance, the normal role of UBE3A in neurons is still unclear. As a step toward deciphering its possible functions, we performed high‐resolution light and electron microscopic immunocytochemistry. We report a broad distribution of UBE3A in neurons, highlighted by concentrations in axon terminals and euchromatin‐rich nuclear domains. Our findings suggest that UBE3A may act locally to regulate individual synapses while also mediating global, neuronwide influences through the regulation of gene transcription. J. Comp. Neurol. 525:1–1, 2017. © 2016 Wiley Periodicals, Inc.

Subcellular organization of UBE3A in neurons.

Ubiquitination regulates a broad array of cellular processes, and defective ubiquitination is implicated in several neurological disorders. Loss of the E3 ubiquitin-protein ligase UBE3A causes Angelman syndrome. Despite its clinical importance, the normal role of UBE3A in neurons is still unclear. As a step toward deciphering its possible functions, we performed high-resolution light and electron microscopic immunocytochemistry. We report a broad distribution of UBE3A in neurons, highlighted by concentrations in axon terminals and euchromatin-rich nuclear domains. Our findings suggest that UBE3A may act locally to regulate individual synapses while also mediating global, neuronwide influences through the regulation of gene transcription. J. Comp. Neurol. 525:233-251, 2017. © 2016 Wiley Periodicals, Inc.

KV1 channels identified in rodent myelinated axons, linked to Cx29 in innermost myelin: support for electrically active myelin in mammalian saltatory conduction.

Saltatory conduction in mammalian myelinated axons was thought to be well understood before recent discoveries revealed unexpected subcellular distributions and molecular identities of the K(+)-conductance pathways that provide for rapid axonal repolarization. In this study, we visualize, identify, localize, quantify, and ultrastructurally characterize axonal KV1.1/KV1.2 channels in sciatic nerves of rodents. With the use of light microscopic immunocytochemistry and freeze-fracture replica immunogold labeling electron microscopy, KV1.1/KV1.2 channels are localized to three anatomically and compositionally distinct domains in the internodal axolemmas of large myelinated axons, where they form densely packed "rosettes" of 9-nm intramembrane particles. These axolemmal KV1.1/KV1.2 rosettes are precisely aligned with and ultrastructurally coupled to connexin29 (Cx29) channels, also in matching rosettes, in the surrounding juxtaparanodal myelin collars and along the inner mesaxon. As >98% of transmembrane proteins large enough to represent ion channels in these specialized domains, ∼500,000 KV1.1/KV1.2 channels define the paired juxtaparanodal regions as exclusive membrane domains for the voltage-gated K(+)conductance that underlies rapid axonal repolarization in mammals. The 1:1 molecular linkage of KV1 channels to Cx29 channels in the apposed juxtaparanodal collars, plus their linkage to an additional 250,000-400,000 Cx29 channels along each inner mesaxon in every large-diameter myelinated axon examined, supports previously proposed K(+)conductance directly from juxtaparanodal axoplasm into juxtaparanodal myeloplasm in mammalian axons. With neither Cx29 protein nor myelin rosettes detectable in frog myelinated axons, these data showing axon-to-myelin linkage by abundant KV1/Cx29 channels in rodent axons support renewed consideration of an electrically active role for myelin in increasing both saltatory conduction velocity and maximum propagation frequency in mammalian myelinated axons.

Hydration status affects osteopontin expression in the rat kidney.

Osteopontin (OPN) is a secretory protein that plays an important role in urinary stone formation. Hydration status is associated with the development of urolithiasis. This study was conducted to examine the effects of dehydration and hydration on OPN expression in the rat kidney. Animals were divided into three groups, control, dehydrated, and hydrated. Kidney tissues were processed for light and electron microscope immunocytochemistry, in situ hybridization, and immunoblot analysis. Dehydration induced a significant increase in OPN protein expression, whereas increased fluid intake induced a decrease in protein expression. Under control conditions, OPN protein and mRNA expression were only detected in the descending thin limb (DTL). Dehydration induced increased expression in the DTL and the development of detectable expression in the thick ascending limb (TAL). In contrast, OPN expression levels declined to less than the controls in the DTL after hydration, while no expression of either protein or mRNA was detectable in the TAL. Immunoelectron microscopy demonstrated that hydration status altered tubular ultrastructure and intracellular OPN expression in the Golgi apparatus and secretory cytoplasmic vesicles. These data confirm that changes in oral fluid intake can regulate renal tubular epithelial cell OPN expression.

Organization of TNIK in dendritic spines.

Tumor necrosis factor receptor-associated factor 2 (TRAF2)- and noncatalytic region of tyrosine kinase (NCK)-interacting kinase (TNIK) has been identified as an interactor in the psychiatric risk factor, Disrupted in Schizophrenia 1 (DISC1). As a step toward deciphering its function in the brain, we performed high-resolution light and electron microscopic immunocytochemistry. We demonstrate here that TNIK is expressed in neurons throughout the adult mouse brain. In striatum and cerebral cortex, TNIK concentrates in dendritic spines, especially in the vicinity of the lateral edge of the synapse. Thus, TNIK is highly enriched at a microdomain critical for glutamatergic signaling.

Metabotropic glutamate receptor 5 shows different patterns of localization within the parallel visual pathways in macaque and squirrel monkeys.

Glutamate is used as an excitatory neurotransmitter by the koniocellular (K), magnocellular (M), and parvocellular (P) pathways to transfer signals from the primate lateral geniculate nucleus (LGN) to primary visual cortex (V1). Glutamate acts through both fast ionotropic receptors, which appear to carry the main sensory message, and slower, modulatory metabotropic receptors (mGluRs). In this study, we asked whether mGluR5 relates in distinct ways to the K, M, and P LGN axons in V1. To answer this question, we used light microscopic immunocytochemistry and preembedding electron microscopic immunogold labeling to determine the localization of mGluR5 within the layers of V1 in relation to the K, M, and P pathways in macaque and squirrel monkeys. These pathways were labeled separately via wheat germ agglutinin–horseradish peroxidase (WGA–HRP) injections targeting the LGN layers. mGluR5 is of interest because it: 1) has been shown to be expressed in the thalamic input layers; 2) appears to be responsible for some types of oscillatory firing, which could be important in the binding of visual features; and 3) has been associated with a number of sensory-motor gating-related pathologies, including schizophrenia and autism. Our results demonstrated the presence of mGluR5 in the neuropil of all V1 layers. This protein was lowest in IVCα (M input) and the infragranular layers. In layer IVC, mGluR5 also was found postsynaptic to about 30% of labeled axons, but the distribution was uneven, such that postsynaptic mGluR5 label tended to occur opposite smaller (presumed P), and not larger (presumed M) axon terminals. Only in the K pathway in layer IIIB, however, was mGluR5 always found in the axon terminals themselves. The presence of mGluR5 in K axons and not in M and P axons, and the presence of mGluR5 postsynaptic mainly to smaller P and not larger M axons suggest that the response to the release of glutamate is modulated in distinct ways within and between the parallel visual pathways of primates.

Phosphorylation of Histone H2AX in the Mouse Brain from Development to Senescence

Phosphorylation of the histone H2AX (γH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.

Hippocampal mossy fiber leu-enkephalin immunoreactivity in female rats is significantly altered following both acute and chronic stress

Research indicates that responses to stress are sexually dimorphic, particularly in regard to learning and memory processes: while males display impaired cognitive performance and hippocampal CA3 pyramidal cell dendritic remodeling following chronic stress, females exhibit enhanced performance and no remodeling. Leu-enkephalin, an endogenous opioid peptide found in the hippocampal mossy fiber pathway, plays a critical role in mediating synaptic plasticity at the mossy fiber-CA3 pyramidal cell synapse. Estrogen is known to influence the expression of leu-enkephalin in the mossy fibers of females, with leu-enkephalin levels being highest at proestrus and estrus, when estrogen levels are elevated. Since stress is also known to alter the expression of leu-enkephalin in various brain regions, this study was designed to determine whether acute or chronic stress had an effect on mossy fiber leu-enkephalin levels in females or males, through the application of correlated quantitative light and electron microscopic immunocytochemistry. Both acute and chronic stress eliminated the estrogen-dependence of leu-enkephalin levels across the estrous cycle in females, but had no effect on male levels. However, following acute stress leu-enkephalin levels in females were consistently lowered to values comparable to the lowest control values, while following chronic stress they were consistently elevated to values comparable to the highest control values. Ultrastructural changes in leu-enkephalin labeled dense core vesicles paralleled light microscopic observations, with acute stress inducing a decrease in leu-enkephalin labeled dense core vesicles, and chronic stress inducing an increase in leu-enkephalin labeled dense-core vesicles in females. These findings suggest that alterations in leu-enkephalin levels following stress could play an important role in the sex-specific responses that females display in learning processes, including those important in addiction.

Fibronectin Antibody Labels Corneal Stromal Collagen Fibrils In Situ Along Their Length and Circumference and Demonstrates Distinct Staining Along the Cell and Stromal Interfaces of Descemet’s Membrane

Purpose/Aim of the study: An immunoperoxidase cytochemical study of fibronectin localization in the rat corneal stroma and Descemet’s membrane was conducted following organ culture to determine whether stromal swelling allowed better primary antibody penetration into the normally tough fibrous corneal stroma.Materials and Methods: Following 24 h organ culture, corneas were fixed in 4% paraformaldehyde, washed and stained overnight at 4 °C in anti-fibronectin followed by washing and incubation in an appropriate secondary antibody and exposure to protein A-HRP. Cytochemical processing was carried out in a DAB-containing medium followed by dehydration and Epon embedding.Results: Observations of the stromal lamellae revealed the presence of a novel punctate staining pattern along the length of the collagen fibrils that extended around the fibril’s circumference. Measurements on the peroxidase reaction product spacing indicated a periodicity of approximately 20.69 ± 3.57 nm along the fibril’s length. Light microscopic immunocytochemistry revealed the presence of fibronectin staining occurred within the endothelial cell layer but only along the DM/stromal interface. Electron microscopic observations however, revealed that fibronectin staining occurred in distinct linear patterns along the length of both the endothelial and stromal DM interfaces.Discussion: Results indicate that organ culture mediated swelling helps facilitate the penetration of primary antibody into the corneal stroma. Observations suggest a novel association exists between fibronectin and stromal collagen fibrils that helps to mediate the arrangement and organization of the stromal extracellular matrix. Results also definitively indicate that fibronectin is deposited along both DM interfaces suggesting that it plays a role in the adhesion of both the endothelial cell layer and stroma to Descemet’s membrane to help maintain the tissue architecture within this region of the cornea.

Catecholaminergic system innervates galanin-immunoreactive neurons in the human diencephalon

Galanin released into the hypophysial portal circulation in the hypothalamus may function as a hypophysiotropic factor regulating the anterior pituitary function or it may function as a neurotransmitter/neuromodulator acting at synaptic sites regulating neuronal activity of many neurons in the brain. Catecholamines (adrenaline, noradrenaline, and dopamine) primarily regulate anterior pituitary functions indirectly via innervating hypophysiotropic neurons. The aim of the present studies was to explore with double-label immunocytochemistry if, as in rodents, catecholamines interact with galanin in the human diencephalon. Due to the long post-mortem period and subsequent lack of optimal preservation of the cell membranes in the brain, electron microscopy could not be employed to show the presence of catecholaminergic-immunoreactive synapses on galanin-immunoreactive neurons. Therefore, we used light microscopic immunocytochemistry and high-magnification microscopy with oil immersion to identify putative juxtapositions between catecholamines and galanin-utilizing antisera against key enzymes of catecholamine synthesis (tyrosine hydroxylase (TH), representing all three catecholamines; dopamine-beta-hydroxylase (DBH), representing noradrenaline; and phenylethanolamine-N-methyltransferase (PNMT), representing adrenaline) and galanin. Our studies show that among the three catecholamines, dopamine is the most abundant and the vast majority of catecholaminergic contacts on galanin-immunoreactive neurons is dopaminergic. The number of DBH-immunoreactive contacts is less and the number of PNMT-immunopositive contacts is negligible. Among the hypothalamic regions, the periventricular region above the infundibulum (infundibular or arcuate nucleus) contained the largest number of contacts. These en passant-type intimate associations between catecholamine- and galanin-immunoreactive neuronal elements may be functional synapses and may provide the morphological basis for the catecholamine-mediated galanin release.

Isolation of a calcium-binding protein of the acrosomal membrane of bovine spermatozoa

The mammalian sperm acrosome reaction is a calcium-dependent exocytotic event characterized by extensive fusion between the plasma and the outer acrosomal membrane. The mechanisms by which elevation of cytosolic calcium initiates the membrane fusion process are not understood and the present study was undertaken to identify calcium-binding proteins in the acrosomal membrane (AM) of bovine spermatozoa. Sperm heads, purified from sonicated spermatozoa, were used to isolate an acrosomal membrane-enriched fraction on Percoll density gradients. Using SDS-PAGE and a 45Ca2+-blot overlay assay, calcium-binding proteins of 64, 45, 43, and 39kDa were identified in the AM enriched fraction. Phase separation analysis with Triton X-114 identified the 64kDa polypeptide as an integral membrane protein. The 64kDa polypeptide was purified and utilized to prepare a polyclonal antiserum. Both light and electron microscopic immunocytochemistry demonstrated that the protein was distributed throughout all domains of the acrosomal membrane. These results identify a 64kDa calcium-binding integral membrane protein of the mammalian acrosome. Its potential function in calcium-dependent membrane fusion events of the acrosome reaction and in fertilization is discussed.

Expression of E-cadherin in pig kidney

E-cadherin is a cell adhesion molecule that plays an important role in maintaining renal epithelial polarity and integrity. The purpose of this study was to determine the exact cellular localization of E-cadherin in pig kidney. Kidney tissues from pigs were processed for light and electron microscopy immunocytochemistry, and immunoblot analysis. E-cadhedrin bands of the same size were detected by immunoblot of samples from rat and pig kidneys. In pig kidney, strong E-cadherin expression was observed in the basolateral plasma membrane of the tubular epithelial cells. E-cadherin immunolabeling was not detected in glomeruli or blood vessels of pig kidney. Double-labeling results demonstrated that E-cadherin was expressed in the calbindin D28k-positive distal convoluted tubule and H+-ATPase-positive collecting duct, but not in the aquaporin 1-positive, N-cadherin-positive proximal tubule. In contrast to rat, E-cadherin immunoreactivity was not expressed at detectable levels in the Tamm-Horsfall protein-positive thick ascending limb of pig kidney. Immunoelectron microscopy confirmed that E-cadherin was localized in both the lateral membranes and basal infoldings of the collecting duct. These results suggest that E-cadherin may be a critical adhesion molecule in the distal convoluted tubule and collecting duct cells of pig kidney.

Adult stem / progenitor cells of the rat thyroid: side population distribution, intermediate filament expression, and long-term in vitro expansion

We recently identified adult stem / progenitor cells in the male rat thyroid, based on expression of the multipotency maker, ATP-binding cassette subfamily G member 2 (ABCG2) (1). To characterize these cells, we have now determined their distribution as a side population of the adult thyroid gland, identified their epithelial vs mesenchymal commitment by the presence of cytoplasmic intermediate filaments, and enriched their number using long-term, in vitro expansion of adherent elements. Sprague-Dawley male rats (50-75 gr) were used as thyroid donors. Following penthobarbital anesthesia rats were thyroidectomised, and primary cells prepared using enzymatic breaking of the gland. After 72 hs in standard monolayer culture, adherent cells were trypsinized, and either incubated for 90 min with the vital dye, Hoechst 33342 (Hch) + the ABCG2 inhibitor, verapamil (VE, 150 mM) followed by cytospin (1300 RPM x 8 min) for single, double, and triple light microscopic immunocytochemistry (IC), or re-seeded (20 x103 / cm2) in monolayer and grown up to 4 months, using a starvation protocol based on a single weekly change of culture medium (low glucose DMEM / 15 % FBS-FHS serum). Co-localization of nuclear Hoch with immunoreactive (IR) ABCG2 (rabbit anti-human polyclonal antiserum, 1:300), IR-cytokeratin (CTK, mAb 1:200), and IR-vimentin (VIM, mAb 1:100) was assessed by the ABC and indirect fluorescence techniques, using DAB, FITC and TRITC as chromogens. Rat kidney, human keratinocyte cell line, NTCT 2544 (courtesy of C. Pellegrini), and primary mouse and human fibroblasts (courtesy of D. Mattioli) were used as positive controls for IC. A consistent increase in Hch-positive nuclei was observed in VE-treated cultures, as opposed to VE-untreaded monolayers. In addition, an inverse staining relationship occurred between nuclear Hch and IR-CTK, as opposed to a direct relationship between nuclear Hch and IR-VIM. Co-localization of IR-ABCG2 with IR-CTK was seen in some cells, whereas that of IR-ABCG2 with IR-VIM was only occasionally detected. Finally, longterm expansion of primary thyrocytes resulted in 30% increase in IR-ABCG2 cells, as opposed to less than 1% IR-ABCG2 elements in standard culture. We conclude that ABCG2-positive cells of the rat thyroid are a side population of stem / progenitor elements, they are primarily committed to the epithelial phenotype, and can be enriched in vitro as adherent cells, suggesting clonal expansion.