Fine structure of male genital tracts of some Acrididae and Tettigoniidae (Insecta: Orthoptera)

A morphological, histological and ultrastructural study was carried out on the spermiducts and seminal vesicles of some species of Acrididae and Tettigoniidae. In all the species examined, the spermiducts and seminal vesicles have a monolayered secretory epithelium. Only the species of Acrididae have the sac with a flattened epithelium. Furthermore, in the most distal tubule region of the seminal vesicles of Eyprepocnemis plorans plorans, a rather characteristic secretory mechanism was found: the cytoplasm of the epithelial cells contained a large vesicle delimited by tightly packed microvilli. Numerous small vesicles open into this large vesicle which gradually dilates to merge with the apical plasma membrane releasing its contents into the lumen. Spermiophagic activity was found in all the species investigated. In the Tettigoniidae, this activity was found only in some epithelial cells of the seminal vesicle wall; in the species of the Acrididae the spermiophagic activity was carried out in the spermiduct lumen by an epithelial‐type cellular group. Spermiophagic activity is discussed as well as its role in the reproduction of these insects.     

Morphology of the Nephron in the Mesonephros of Bufo bufo (Amphibia,Anura, Bufonidae)

The present study deals with the morphology and ultrastruclure of the nephron in the mesonephros of the toad, Bufo bufo (Linnaeus, 1758). Based on serial sections in paraffin, Araldite and Epon, the position of the different segments of the nephron within the kidney tissue was determined, and a nephron subsequently reconstructed. The nephron consists of the following parts: Malpighian corpuscle, neck segment, proximal tubule, intermediate segment, early distal tubule, late distal tubule and collecting tubule. The late distal tubule was subdivided into three morphologically different sections. The total number of nephrons in the toad mesonephros was estimated at 6000 units. The length of the segments in the reconstructed nephron was calculated. The cytology of the epithelial cells constituting the segments was described using transmission and scanning electron microscopy. Heterocellularity was found in the late distal tubule section I and III and in the collecting tubule. The proportional distribution and number of intercalated (mitochondria-rich) cells in the late distal tubule and collecting tubule was calculated. Only one morphological type of intercalated cell could be distinguished. Late distal tubules were removed from fresh Bufo kidneys for preliminary studies of the intercalated cells with Nomarski optics.     

Quantitation of glucose-6-phosphate dehydrogenase activity in cortical fractions of the nephron in sodium-depleted and sodium-loaded rabbits

Summary Glucose-6-phosphate dehydrogenase activity was measured quantitatively in isolated cortical fractions of the nephron in sodium-depeleted and sodium-loaded rabbits. The samples consisted of isolated fractions of macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. In sodium-depleted rabbits enzyme activity was identical to that of normal rabbits. In sodium-loaded rabbits a significant decrease in enzyme activity was found in the macula densa and proximal convoluted tubule. However, using conventional histochemical incubation methods semiquantitative estimation of glucose-6-phosphate dehydrogenase showed a slight decrease in enzyme activity in the macula densa and distal convoluted tubule, and a slight increase in the proximal convoluted tubule during sodiumdepletion. During sodium-load a pronounced decrease in enzyme activity was seen in the macula densa and distal convoluted tubule. These results show that semiquantitative histochemical evaluation of changes in enzyme activity is less reliable than the more precise quantitative method especially when there are only slight changes in enzyme activity. Only where there were marked changes in histochemical enzyme activity might the results of quantitative and semiquantitative methods be in accord.     

Developmental origin of segmental differences in the leech ectoderm: survival and differentiation of the distal tubule cell is determined by the host segment

The body plan of the adult leech is metameric, with each hemisegmental complement of ectodermal and mesodermal tissues being produced from a set of seven serially repeated embryonic blast cells. Previous studies have shown that homologous o blast cells give rise to an almost identical complement of descendant cells in each of the 21 abdominal segments, but that one o blast cell derivative--the distalmost cell of the nephridial tubule--is only present in 15 abdominal segments in the mature leech. Here we show that all o blast cells generate a presumptive distal tubule cell and that this cell migrates to its normal position in all abdominal segments. However, in segments which normally do not contain the mesodermal portion of the nephridium, the distal tubule cell dies before undergoing its terminal morphological differentiation. To ascertain whether the fate of the distal tubule cell is determined by its lineage history or by the segmental environment into which it is born, we utilized a previously described procedure for altering the segmental register between different embryonic cell lines. This procedure allowed us to effectively transplant o blast cells into more posterior segments prior to the cell divisions which generate their descendant clones. The results indicate that the survival or death of the distal tubule cell is determined by the identity of the host segment and that a given distal tubule cell could be effectively murdered or rescued by slipping its blast cell precursor into an appropriate segment. These findings suggest that the segment-specific pattern of distal tubule cell survival is not inherent to the O cell line, but arises from interactions with surrounding tissues.     

Oxidative enzymes in the urinary apparatus of several marine fishes

Summary The distribution and activities of several oxidative enzymes in the urinary apparatus of seven marine fish species (hagfish, lesser spotted dogfish, electric ray, herring, marine catfish, cod, sea-horse) have been studied. Species were selected from three main taxonomic groups: Cyclostomata, Elasmobranchii and Teleostei. Distinctly positive enzyme reactions were found in the tubular elements of the kidney and the collecting duct-archinephric duct system, with the exception of the generally weak staining intensities for NADP-liked malate dehydrogenase. In the proximal tubule segment the second, more distal part (PII) reacted, in general, very strongly when compared with the first proximal part (PI). If present, the distal tubule in teleosts showed only weak reactions, while this segment in elasmobranchs exhibited moderate to strong enzyme activities. In the epithelial cells of the collecting tubule-collecting duct system stronger reactions were confined to the glomerular teleost species, the corresponding part of the elasmobranch kidney showing weak staining intensities. In the urinary duct system distinctly positive enzyme reactions were only to be found in the archinephric duct of the teleost species, except forPlotosus. The ureters of the elasmobranchs exhibited weak enzyme activities throughout.The enzyme patterns of the various types of urinary tubules and ducts are compared with observations from several morphological and physiological studies. The histochemical findings are discussed in relation to corresponding investigations of fresh water fishes and problems arising from phylogenetic divergence of marine fish groups.     

Porcine myosin-VI: characterization of a new mammalian unconventional myosin

We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC- PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.     

Predicting the functionally distinct residues in the heme,cation, and substrate-binding sites of peroxidase from stress-tolerant mangrove specie, <Emphasis Type="Italic">Avicennia marina</Emphasis>

Recent work was conducted to predict the structure of functionally distinct regions of Avicennia marina peroxidase (AP) by using the structural coordinates of barley grains peroxidase as the template. This enzyme is utilized by all living organisms in many biosynthetic or degradable processes and in defense against oxidative stress. The homology model showed some distinct structural changes in the heme, calcium, and substrate-binding regions. Val53 was found to be an important coordinating residue between distal calcium ion and the distal heme site while Ser176 is coordinated to the proximal histidine through Ala174 and Leu172. Different ionic and hydrogen-bonded interactions were also observed in AP. Analyses of various substrate–enzyme interactions revealed that the substrate-binding pocket is provided by the residues, His41, Phe70, Gly71, Asp138, His139, and Lys176; the later three residues are not conserved in the peroxidase family. We have also performed structural comparison of the A. marina peroxidase with that of two class III salt-sensitive species, peanut and soybean. Four loop regions were found to have largest structural deviation. The overall protein sequence was also analyzed for the presence of probable post-translational modification sites and the functional significance of these sites were outlined.     

Carbonic anhydrase activity in kidney and lower intestine of the European starling

A histochemical investigation of kidney and lower intestine of the European starling (Sturnus vulgaris) shows no carbonic anhydrase activity in proximal convoluted tubules, although activity is seen in similarly prepared sections of rat proximal tubules. Early distal tubule cells in the starling are stained throughout the cytoplasm and at the apical and highly infolded basolateral membranes. Late distal tubules lose apical activity and have reduced basolateral infolding, resulting in less intense staining. Darkly stained intercalated cells appear in the connecting tubules and cortical collecting ducts. Both of these segments also show intense basolateral staining. Medullary cones of the starling are highly organized, with central zones containing unstained thin descending limbs of loops of Henle, surrounded by both medullary collecting ducts with only scattered cells staining for enzyme, and by thick ascending limb segments. The latter contain many uniformly stained cells intermingled with occasional unstained cells. Scattered cells of the starling colonic villi demonstrate intense apical brush border membrane staining as well as cytoplasmic staining. Cells lining the cloaca stain less intensely. A biochemical assay for carbonic anhydrase was used to quantify enzyme activity in these tissues. Starling kidney contained 1.96 ± 0.33 (mean ± SEM) enzyme units/mg protein, less than half the activity seen in rat kidney. Stripped colonic epithelium contained 0.66 ± 0.15 enzyme units/mg protein. These quantitative results correlate well with the interpretations derived from the histochemical observations. The lack of proximal tubule carbonic anhydrase activity suggests that the avian kidney relies more on distal nephron segments to achieve net acidification of the urine.     

Corticosteroid induction of renal and intestinal K+-dependentp-nitrophenylphosphatase in young and adult rats

Summary The post-natal development of the K⁺-dependentp-nitrophenylphosphatase (K-NPPase) activity of the Na, K-ATPase complex and its regulation by corticosteroids was studied in renal and intestinal epithelia of the rat using thep-nitrophenylphosphatecerium capture method. The distribution of the phosphatase was analysed in detail in the renal epithelia of the medullary thick ascending limb of Henle's loop and distal convoluted tubule and in the surface epithelial cells of the distal colon. The convoluted tubule and Henle's loop segments showed a stronger reaction for K-NPPase than the colon epithelium both in adult and young animals (suckling and weanling pups). The intensity of staining rose progressively in all three epithelia during early postnatal development and reached the highest levels during the weaning period and in adulthood. The most distinct change was observed between days 10 and 16. Adrenalectomy significantly reduced the density of the final reaction product in weanling and adult rats. Replacement hormone therapy of adrenalectomized weanling rats with the glucocorticoid dexamethansone restored the K-NPPase activity in the two renal epithelia, whereas the mineralocorticoid deoxycorticosterone acetate had no effect on the activity in the medullary thick ascending limb, a very slight effect in distal convoluted tubules, and a strong effect on the distal colon epithelial activity. The observed small effect of the mineralocorticoid in distal convoluted tubule activity may reflect a cross-over into glucocorticoid receptors. We conclude that the postnatal development of Na,K-ATPase is regulated by glucocorticoids in nephron epithelia and predominatly by mineralocorticoids in the surface enterocytes of the distal colon.     

Radiosensitivity of mammalian cell lines engineered to overexpress cytosolic glutathione peroxidase

Reactive oxygen species are believed to be involved in radiation lethality. Glutathione peroxidase is an intracellular enzyme with antioxidant functions. To determine whether increasing the cellular antioxidant capacity can confer radiation resistance, the effect of overexpression of glutathione peroxidase on radiosensitivity was determined in two different cell types. An expression construct including the bovine cytosolic glutathione peroxidase cDNA was used to overexpress this enzyme in cells of the human lymphoblast cell line Sup-T1 as well as the Chinese hamster ovary cell line AA8. Supplementation of the culture media with 30 nM sodium selenite was included to obtain optimal glutathione peroxidase activity. Northern blot analysis confirmed the presence of the construct mRNA, and a standard coupled spectrophotometric assay demonstrated significantly increased glutathione peroxidase activity in the transfected cell lines. An approximately 8-fold increase was found in the Sup-T1 cells, and an approximately 30-fold increase was obtained in the Chinese hamster ovary AA8 cells. Clonogenic survival was assayed in the overexpressing cells and compared to that in control cells transfected with vector alone. Despite significantly increased glutathione peroxidase activity, no observable radioprotection was conferred in either of the two cell lines studied, indicating that increased glutathione peroxidase activity is insufficient to confer radioresistance in the two cell types examined. These data are discussed in the context of using antioxidants as adjuncts to clinical radiotherapy.     

Structure of the kidney in the crab-eating frog, Rana cancrivora

The structure of the nephron in the ranid frog, Rana cancrivora, was studied by light and electron microscopy. This frog is the only amphibian species to live in mangrove swamps of very high salinity. The nephron consists of the following parts: renal corpuscle, ciliated neck segment, proximal tubule, ciliated intermediate segment, distal tubule, connecting tubule, and collecting duct. The distal tubule is located in the ventromedial region of the kidney, and the other tubules are situated in the dorsolateral region. Renal corpuscles are found between the two regions. Some renal corpuscles have a wide Bowman's space because of the small glomerulus within them. The proximal tubules are composed of columnar cells with a dense luminal brush border of long microvilli and numerous apical vesicles and vacuoles. The initial part of the distal tubule consists of heavily interdigitated cells, characterized by a very regular palisade arrangement of mitochondria. In the terminal part of the distal tubule, shorter mitochondria of the infolding cells are situated irregularly around the nucleus. The connecting tubule consists of principal cells and canaliculus cells. The collecting duct consists of columnar or cuboidal cells; cytoplasmic organelles are relatively sparse. The canaliculus cells are intercalated between principal cells from the terminal distal tubule to the proximal part of the collecting duct. Our findings indicate that the kidney of R. cancrivora is structurally similar to kidneys of other amphibians. These findings are discussed with regard to probable correlations between ultrastructure and function in R. cancrivora.     

A comparative morphological and enzyme histochemical study on blood cells of the freshwater snails Lymnaea stagnalis,Biomphalaria glabrata,and Bulinus truncatus

The morphology and ultrastructure of the blood cells of the freshwater snails Lymnaea stagnalis, Biomphalaria glabrata, and Bulinus truncatus were studied. By performing in vitro experiments and enzyme histochemical studies, special attention was paid to the role of the blood cells in phagocytosis of foreign particles. No fundamental differences were found in the ultrastructure, lysosomal enzyme contents, and phagocytic capacities of the blood cells of these species. It is concluded that only one type of blood cell, the amoebocyte, exists in the freshwater snails. Amoebocytes constitute a morphologically and functionally heterogeneous population of cells, ranging from round (electron-dense) cells with the morphological characteristics of young cells to highly phagocytic spreading cells with a prominent lysosomal system. In addition to acid phosphatase, nonspecific esterase and peroxidase were found within the lysosomes. The presence of enzyme activity in the RER and the Golgi bodies indicates that amoebocytes are able to synthesize lysosomal enzymes continuously.     

Ultrastructure of maxillary gland of Antrobathynella stammeri (Syncarida,Malacostraca)

The maxillary gland of the highly adapted stygobiont species, Antrobathynella stammeri (Bathynellacea, Syncarida), consists of an end sac, an excretory tubule, and a terminal duct. No valve was found. The excretory tubule forms a loop extending back into the fourth thoracic segment. The end sac is composed of five typical podocytes. Ultrastructurally and functionally, two cell types characterize, respectively, proximal and distal sections of the excretory tubule. Epithelial cells are covered with extremely broad (up to 0.4 μm) microvilli. A basal labyrinth was not seen. Therefore, it is unlikely that the maxillary gland is able to produce a hypoosmotic urine necessary for freshwater animals. Tubule cells can be surrounded by parenchymal cells that produce numerous vesicles, suggesting possible physiological interactions between tubule cells and parenchyma. The ectodermal terminal duct is lined with cuticle and is differentiated into a pulsatile body consisting of two interconnected ampules. The first functions as a bladder. The second ampule, under muscular control, excretes the urine. Pulsatile body, looping tubule, and broad microvilli appear to be distinctive features of the bathynellacid excretory organ. © 1996 Wiley-Liss, Inc.     

Fine structure of the chemoreceptor sensillum in Limulus

Each chemoreceptor sensillum of Limulus polyphemus consists of 6–15 bipolar neurosensory cells with distal processes confined within a single cuticular tubule as they extend to the outside environment. The cuticular tubule, which is enveloped by the cuticulo-tubal cell, opens proximally into a fluid-filled extracellular space through which the dendrite passes before entering the cuticular tubule. Between the neurosensory cells are one to three microvillar cells also exposed to the extracellular space. This space is enclosed by a sheath cell extending proximally from the inner opening of the cuticular tubule and enveloping the proximal portions of the dendrites, the distal portions of the microvillar cells, as well as the distal portion of some neurosensory cell bodies. Most of the remaining portions of the neurosensory cells and microvillar cells are enveloped by neuroglia. Tight junctions occur between the distal portions of the dendrites in or near the cuticular tubule. Each dendrite has a cilium-like segment located where it traverses the extracellular space with a 9 + 0 pattern of fibers. Septuplelayered junctions occur among the proximal portions of some dendrites and some neurosensory cell bodies of the same sensillum. The subjacent processes of the sensillum frequently course proximally as isolated axons before joining nerve bundles. In the chilarial and gnathobasal chemoreceptors these nerve bundles course proximally to neuropile clumps of a peripheral nerve plexus. The presence of numerous synaptic vesicles in the neuropiles suggests that chemical transmission may occur among “en passant” synapses formed by the axons. Proximally the neuropiles are joined to the central nervous system by relatively long nerves.     

Carbonic anhydrase in the monkey kidney

The distribution of carbonic anhydrase in the kidney of the cynomolgus monkey was studied by the histochemical method of Hansson. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed high enzyme activity at the brush border and the basolateral membranes and the cytoplasm. Straight proximal tubules were less intensely stained. In nephrons with long loops of Henle, the descending thin limb contained weak enzyme activity, whereas the ascending thin limb was inactive. The thick limb of Henle's loop displayed most enzyme activity at the luminal cell border. In distal convoluted tubules enzyme activity was restricted to the basal part of the cells. In the late distal tubule, intercalated cells appeared among the "ordinary" distal cells and contained abundant cytoplasmic enzyme. Many intensely stained intercalated cells were also found in the cortical and outer medullary segments of the collecting duct, intermingled with more weakly stained chief cells. In the inner medullary segment of the collecting duct, enzyme activity gradually disappeared. Many capillaries were clearly stained for enzyme activity. The capillary staining apparently varied with that of the kidney tubules; virtually all capillaries in the cortex, but very few in the inner medulla, were stained. The distribution of carbonic anhydrase in the kidney tubules of the monkey is very similar to that in man and in the rat, but the primate kidney differs from the rat kidney by the presence of capillary enzyme activity. The functional importance of this difference is not clear at present.     

Diffusion artefacts and tissue fixation in thyroperoxidase cytochemistry

The distribution of endogenous peroxidase activity in rat, mouse and human thyroid follicle cells was studied with electron microscopic cytochemistry after incubation in 3-3'-diaminobenzidine (DAB). In all three species enzyme activity was found at the apical plasma membrane (facing the follicle lumen) as well as in intracellular compartments. The enzyme activity in the apical plasma membrane was more sensitive to changes in fixation conditions than the activity in intracellular compartments. Under optimal conditions more than 90% of the follicle cells in normal rat thyroids displayed a cytochemical reaction at the apical plasma membrane. In all three species the reaction product at the apical plasma membrane formed a gradient which extended into the colloid which otherwise was unreactive. Evidence obtained indicated that this gradient was not due to the presence of soluble peroxidase in the lumen but most likely signified the diffusion of the reaction product from the membrane-bound enzyme.     

Quantitative measurement of glucose-6-phosphate dehydrogenase in cortical fractions of the rabbit nephron

A quantitative fluorimetric method is described for estimating the activity of glucose-6-phosphate dehydrogenase in isolated fractions of rabbit nephron from the superficial part of the renal cortex: macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. The mean activity in the macula densa region was 2.5 X 10(-18) mol/micrometers 3/min, which was about twice the mean activity of the proximal and distal tubular cells and four times that of the glomeruli. As glucose-6-phosphate dehydrogenase is located in the cytoplasm, the average cytoplasmic enzyme activity of the different tubular cells was calculated: macula densa activity was 4.0 X 10(-18) mol/micrometers 3/min whilst proximal tubular cells showed about a third, and distal tubular cells about a quarter of this activity.     

Ultrastructure of the tubular nephron of the lizard Podarcis (= Lacerta) Taurica

The proximal, intermediate, and distal convoluted tubules of the neprhon of Podarcis (= Lacerta) taurica were examined by electron microscopy. Proximal tubule cells have large, apical cytoplasmic protrusions and microvilli interpreted to function in urate secretion. Adjacent cells are bound apically by tight junctions and desmosomes but interdigitate in their basal region. This situation is repeated in the other tubules with significant differences in intercellular space width. The basal surfaces bear numerous cytoplasmic processes. The intermediate tubule has proximal and distal segments each with dark, ciliated, and light cells, the cuboidal dark cells with dense cytoplasm constituting the main bulk of the wall. As the cells of the proximal and distal segments resemble those of the proximal and distal convoluted tubules, respectively, the intermediate tubule is considered as a transition region. The ciliated cell body has two broad processes extending from the lumen, one to the basement membrane and one to a foot process of a light cell. The light cell is surrounded by dark and ciliated cells. It does not reach the lumen, but contacts the basement membrane through a process running below a ciliated cell to form a mushroom-shaped structure in tubule cross-section, the light cell process forming the stalk and a ciliated cell the cap. The cilia probably propel the glomerular filtrate towards the distal convoluted tubule. This latter tubule has initial, middle, and terminal zones, all nonciliated but with different lumen widths and cell shapes.     

The pelvic kidney of male Ambystoma maculatum (Amphibia,urodela, ambystomatidae) with special reference to the sexual collecting ducts

This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct, synthesizes neutral carbohydrates and proteins, and is also lined by apical ciliated cells intercalated between secretory cells. Although functional aspects associated with the morphological variation along the length of the proximal portions of the nephron have been investigated, the role of a highly secretory collecting duct has not. Historical data that implicated secretory activity concordant with mating activity, and similarity of structure and chemistry to sexual segments of the kidneys in other vertebrates, lead us to believe that the collecting duct functions as a secondary sexual organ in Ambystoma maculatum. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.