Nutritional cellular biomarkers in early life stages of fish

Histological (tissular and cellular) indices have a tradition of determining the nutritional condition of fish both in the laboratory and in the wild. The assessment of condition by means of microscopical methods is probably the mos accurate indicator of nutritional status during the early life stages of fish. This success is partly attributable large amount of information that can be derived from their study and because they are thought to be the only true starvation indices. The technique usually consists of the examination of cells and organs and the establishment of a grading system based on the presence/absence of standardised biomarkers. Each organ is examined, and the cellular aspect or tissular cohesion is evaluated qualitatively and even quantitatively in order to obtain a measure of the general condition of a larva. The literature indicates that there are certain tissular and cellular responses to food availability and quality, particularly in the digestive and muscular tissues, which are common to most teleost fish larvae. These responses, which are independent of water temperature, can be used for assessing fish larvae nutritional condition. In this regard, the microscopical organization of the liver hepatocytes, the intestinal mucosa, the exocrine pancreas and the muscular fibers, which are generally used as target tissues and organs to assess the nutritional condition of fish larvae, is deeply reviewed. The advantages and disadvantages of the use of different cellular biomarkers of effect are discussed considering different conditions.     

Fungal and algal gene expression in early developmental stages of lichen-symbiosis

How plants and microbes recognize each other and interact to form long-lasting relationships remains one of the central questions in cellular communication. The symbiosis between the filamentous fungus Cladonia grayi and the single-celled green alga Asterochloris sp. was used to determine fungal and algal genes upregulated in vitro in early lichen development. cDNA libraries of upregulated genes were created with suppression subtractive hybridization in the first two stages of lichen development. Quantitative PCR subsequently was used to verify the expression level of 41 and 33 candidate fungal and algal genes respectively. Induced fungal genes showed significant matches to genes putatively encoding proteins involved in self and non-self recognition, lipid metabolism, and negative regulation of glucose repressible genes, as well as to a putative d-arabitol reductase and two dioxygenases. Upregulated algal genes included a chitinase-like protein, an amino acid metabolism protein, a dynein-related protein and a protein arginine methyltransferase. These results also provided the first evidence that extracellular communication without cellular contact can occur between lichen symbionts. Many genes showing slight variation in expression appear to direct the development of the lichen symbiosis. The results of this study highlight future avenues of investigation into the molecular biology of lichen symbiosis.     

Nongranular vasopressin synthesis and transport in early stages of rehydration

Summary The vasopressin system of the rat was examined in the course of the first 12 h of rehydration after prolonged thirst at light and electron microscopic levels and by use of the peroxidase anti-peroxidase (PAP) method. Light microscopically, the median eminence was the only part of the system that not only displayed distinct differences between animals of different rehydration times but also showed a characteristic pattern of immunohistochemical reactivity in its rostro-caudal distribution. Ultrastructurally, in the perikarya a maximal labeling of the rough endoplasmic reticulum was observed after 2 h of rehydration, whereas an extensive labeling of the enlarged Golgi zones was attained after 4 h of resupplying water. A labeling of the intercellular clefts in the basal glial labyrinth of the supraoptic nucleus (and to a lesser degree in the subependymal neuropil adjacent to the paraventricular nucleus) was increased 30 min after the onset of drinking, as compared with water-deprived animals; it decreased slightly after 12 h of rehydration. The filling of the swollen fibers by increasing amounts of labeled axoplasmic reticulum, evident in the nuclear areas already after 30 min of water supply, begins in the median eminence after 2 h of rehydration and is fully developed after 4 and 8 h. Corresponding results hold true for the neural lobe but are somewhat delayed in comparison to the findings in the median eminence. The discussion considers (i) synthesis and transport of nongranular vasopressin within the axoplasmic reticulum, and (ii) release not only from the neural lobe but also from the nuclear areas and from the fibers of the median eminence.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and Stiftung Volkswagenwerk     

Blood supply of the compact and spongy myocardium of fish, amphibia and reptiles]

Coronal arteries were injected with lead carbonate suspension and with Indian ink and cleared preparations 150--300 mkm thick were made in 195 hearts of fish, amphibians and reptiles and studied roentgenographically. It was stated that in Chondrichthyes (shark, skate) and in Chondrostei (beluga, stellate sturgeon, sturgeon), as well as in alligator both compact and spongy myocardium of the cardiac ventricle possess blood vessels. In teleostei, amphibians and reptiles (except alligator) spongy myocardium is avascular and receives its nutrition from the ventricle. In view of the data on the presence of blood vessels in the spongy myocardium in some vertebrates, it is impossible to accept the theory suggested by Grant and Regnier according to which vessels in the heart walls appear only in connection with compactization of the myocardium. Vascularization of the spongy myocardium is closely connected with oxygen saturation of the blood flowing through the heart. When this saturation is not satisfactory, the spongy myocardium has blood vessels. In alligator, vascularization of the spongy myocardium is connected with the fact that the heart has four chambers and there are arterial and venous blood streams.     

Detecting physiological and pesticide-induced apoptosis in early developmental stages of invasive bivalves

In this study, embryos and early larval stages of two invasive bivalves, the Mediterranean mussel (Mytilus galloprovincialis) and zebra mussel (Dreissena polymorpha), were assayed for physiological apoptosis and stress-induced apoptosis post-exposure to a molluscicide, Bayluscide^®. Physiological apoptosis was measured at 4-h intervals and Bayluscide^® exposures were 4 h and 24 h beginning at both 30 min post-fertilization and at 24 h post-fertilization. Apoptosis was detected by the TUNEL assay, and apoptotic values were compared for the different stages present. The two bivalves tested for apoptotic cells displayed variations in physiological and stress-induced apoptotic values despite having the same life stage transitions. However, in the majority of exposures, we found that as pesticide concentrations increased, the apoptotic values decreased in the highest concentrations. We suggest that this change to apoptotic inhibition indicates that there is a threshold at which apoptosis can no longer be used as a protective measure against damaged cells. These concentrations that cause changes in apoptotic pattern should be further tested for long-term effects on reproduction and survival. By detecting sub-lethal concentrations that may impair recruitment to the population and the continuation of the life cycle, chemical control of invasive bivalves could be targeted to early life stages and to reduce potential environmental impacts resulting from the higher concentrations required to control adults.     

Intermediate filament protein expression in early developmental stages of the mouse

Expression patterns of intermediate filament proteins have been studied during early mouse embryo development. For this purpose, pre-implantation embryos at different stages of development after in vitro fertilization were studied using antibodies to cytokeratins, vimentin and lamins, using the indirect immunofluorescence assay. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies showed that, although the embryos grew in culture, vimentin could not be detected in a filamentous organization. Immunofluorescence for cytokeratins was only positive from the 8-cell stage onwards. In the morula stage an increased level of cytokeratin expression was observed with a transitional staining pattern, combining a filamentous and a diffuse occurrence. In the blastocyst stages profound cytokeratin filaments were seen in trophoblast cells but not in the inner cell mass. When the cytokeratin subtypes were analysed separately, it became apparent that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage and showing a filamentous distribution, were not found as prominently as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected in all developmental stages examined, as a faintly reactive nuclear lamina. In blastocysts both lamin types were detected in trophoblast as well as in inner cell mass.     

A progestin and an estrogen regulate early stages of oogenesis in fish

Using two species of teleost fish, Japanese huchen (Hucho perryi) and common carp (Cyprinus carpio), we investigated whether sex steroids are involved in early oogenesis in vitro. Ovarian fragments were cultured to examine the effects of a progestin, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP), and an estrogen, estradiol-17 beta (E2). DHP and E2 significantly promoted DNA synthesis in ovarian germ cells, as judged by 5-bromo-2-deoxyuridine (BrdU) incorporation into these cells. Furthermore, to detect the initiation of the first meiotic division of early oogenesis, we assessed ultrastructurally the occurrence of synaptonemal complexes (SCs) and analyzed by immunohistochemistry the expression of a meiosis-specific marker, Spo11. In huchen, a higher percentage of oocytes with SC was seen in DHP-treated ovarian fragments than in control or E2-treated ovarian fragments. Spo11 was expressed in germ cells after DHP treatment of carp ovarian explants. These data suggest that the progression of germ cells through early oogenesis involves two sex steroids: E2, which acts directly on oogonial proliferation, and DHP, which acts directly on the initiation of the first meiotic division of oogenesis.     

Cell signaling and ion transport across the fish gill epithelium

A large array of circulating and local signaling agents modulate transport of ions across the gill epithelium of fishes by either affecting transport directly or by altering the size and distribution of transporting cells in the epithelium. In some cases, these transport effects are in addition to cardiovascular effects of the same agents, which may affect the perfusion pathways in the gill vasculature and, in turn, affect epithelial transport indirectly. Prolactin is generally considered to function in freshwater, because it is the only agent that allows survival of some hypophysectomized fish species in freshwater. It appears to function by either reducing branchial permeability, Na,K-activated ATPase activity, or reducing the density of chloride cells. Cortisol was initially considered to produce virtually opposite effects (e.g., stimulation of Na,K-activated ATPase and of chloride cell size and density), but more recent studies have found that this steroid stimulates ionic uptake in freshwater fishes, as well as the activity of H-ATPase, an enzyme thought to be central to ionic uptake. Thus, cortisol may function in both high and low salinities. Growth hormone and insulin-like growth factor appear to act synergistically to affect ion regulation in seawater fishes, stimulating both Na,K-activated ATPase and Na-K-2Cl co-transporter activity, and chloride cell size, independent of their effects on growth. Some of the effects of the GH-IGF axis may be via stimulation of the number of cortisol receptors. Thyroid hormones appear to affect seawater ion regulation indirectly, by stimulating the GH-IGF axis. Natriuretic peptides were initially thought to stimulate gill ionic extrusion, but recent studies have not corroborated this finding, so it appears that the major mode of action of these peptides may be reduction of salt loading by inhibition of oral ingestion and intestinal ionic uptake. Receptors for both arginine vasotocin and angiotensin have been described in the gill epithelium, but their respective roles and importance in fish ion regulation remains unknown. The gill epithelium may be affected by both circulating and local adrenergic agents, and a variety of studies have demonstrated that stimulation of alpha-adrenergic versus beta-adrenergic receptors produces inhibition or stimulation of active salt extrusion, respectively. Local effectors, such as prostaglandins, nitric oxide, and endothelin, may affect active salt extrusion as well as gill perfusion. Recent studies have suggested that the endothelin inhibition of salt extrusion is actually mediated by the release of both NO and prostaglandins. It is hoped that modern molecular techniques, combined with physiological measurements, will allow the dissection of the relative roles in ion transport across the fish gill epithelium of this surprisingly large array of putative signaling agents.     

The role of volume-sensitive ion transport systems in regulation of epithelial transport

This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascites tumour cells and NIH-3T3 cells to elucidate osmotic regulation of salt transport in epithelia. Using the intestine of the European eel (Anguilla anguilla) (an absorptive epithelium of the type described in the renal cortex thick ascending limb (cTAL)) we have focused on the role of swelling-activated K+- and anion-conductive pathways in response to hypotonicity, and on the role of the apical (luminal) Na+-K+-2Cl- cotransporter (NKCC2) in the response to hypertonicity. The shrinkage-induced activation of NKCC2 involves an interaction between the cytoskeleton and protein phosphorylation events via PKC and myosin light chain kinase (MLCK). Killifish (Fundulus heteroclitus) opercular epithelium is a Cl(-)-secreting epithelium of the type described in exocrine glands, having a CFTR channel on the apical side and the Na+/K+ ATPase, NKCC1 and a K+ channel on the basolateral side. Osmotic control of Cl- secretion across the operculum epithelium includes: (i) hyperosmotic shrinkage activation of NKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC by hypotonic cell swelling and a protein phosphatase, and (iii) a protein tyrosine kinase acting on the focal adhesion kinase (FAK) to set levels of NKCC activity.     

Comparative organ differentiation during early life stages of marine fish

The basic developmental mechanisms of teleosts are similar, but there are differences with respect to the timing of developmental events. These events are controlled by genetic and environmental factors. Direct comparisons of organogenesis are complicated due to large variations in egg sizes and incubation temperatures between species. But in general, cultivated small marine pelagic fish larvae originating from rather small eggs (like gadoids, flatfishes, sparids) hatch with a relatively large yolk sac, a larval finfold and subdermal space and under-developed organs. Developmental status at hatch differs between species and the duration of the yolk sac period varies. Main organs and organ systems become functional by first feeding and differentiate during the larval stage and metamorphosis. Species developing directly via large yolk-rich eggs and a long incubation period have a juvenile like morphology and organ functionality at first feeding, sometimes immediately after hatch (like wolffishes). Histomorphological and cell- or organ functional studies of developing embryos and larvae of cultivated species constitute basic information for understanding species-specific events, of utmost importance for improving production protocols. Information is still lacking on early functionality of endocrine and immunocompetent tissues and organs, areas that deserve future focus.     

Inducibility of life history changes by fish kairomone in various developmental stages of Daphnia

Life history changes in Daphnia galeata as a response to part-timeexposure to fish-exuded chemical (FEC) were studied in laboratoryexperiments. Exposure to FEC during one of the juvenile instarsdid not induce significant changes in life history parametersin D.galeata primiparae. Exposure in the first two instars injuvenile development induced earlier maturation, smaller primiparaeand the production of smaller eggs. Exposure in the third andfourth juvenile instars resulted in smaller primiparae withsmaller eggs. The adult daphnids, when exposed to FEC, reducedsomatic growth and produced more and smaller offspring. Resultsof other experiments presented here indicate that the perceptionof fish chemical signal by daphnids is not mediated by the ingestionof a chemically modified diet.     

Defining early human NK cell developmental stages in primary and secondary lymphoid tissues

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56^brightCD16^+/− and CD56^dimCD16⁺ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body.     

Some antioxidant and senescence parameters of broccoli as related to its developmental stages

In broccoli heads grown in spring and in autumn, and harvested at four various developmental stages antioxidant ability as well as certain indices of plant senescence were investigated. Development of broccoli heads was accompanied by losses of chlorophyll and ascorbic acid. At pre-florescence stage accumulation of soluble phenolics was observed. Activity of peroxidase in the initial stage of growth increased significantly and its high level was mantained during the whole growing period. The profound evolution of ethylene was manifested by plants of the pre-florescence stage. In broccoli grown in spring the higher contents of chlorophyll and phenolics in comparison with the autumn experiment were observed, as well as slightly higher activities of peroxidase and catalase. Total antioxidant activity was considerably higher in spring than in autumn.     

Analysis of cerebro-spinal fluid protein composition in early developmental stages in chick embryos

Foetal cerebro-spinal fluid (CSF) has a very high protein concentration when compared to adult CSF, and in many species five major protein fractions have been described. However, the protein concentration and composition in CSF during early developmental stages remains largely unknown. Our results show that in the earliest stages (18 to 30 H.H.) of chick development there is a progressive increase in CSF protein concentration until foetal values are attained. In addition, by performing electrophoretic separation and high-sensitivity silver staining, we were able to identify a total of 21 different protein fractions in the chick embryo CSF. In accordance with the developmental pattern of their concentration, these can be classified as follows: A: high-concentration fractions which corresponded with the ones described in foetal CSF by other authors; B: low-concentration fractions which remained stable throughout the period studied; C: low-concentration fractions which show changes during this period. The evolution and molecular weight of the latter group suggest the possibility of an important biological role. Our data demonstrate that all the CSF protein fractions are present in embryonic serum; this could mean that the specific transport mechanisms in neuroepithelial cells described in the foetal period evolve in very early stages of development. In conclusion, this paper offers an accurate study of the protein composition of chick embryonic CSF, which will help the understanding of the influences on neuroepithelial stem cells during development and, as a result, the appropriate conditions for the in vitro study of embryonic/foetal nervous tissue cells.     

Antioxidant enzymatic systems in pigment tissue of amphibia

Superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities in pigmented and unpigmented liver tissues of frog and albino rat, respectively, were studied. Our results show that pigmented tissue is lacking in manganese superoxide dismutase activity and that the main enzymatic activity utilized in the cytosol by pigmented cells to reduce the hydrogen peroxide to water is represented by catalase; on the contrary, for the same reaction, the cells of albino rat liver primarily utilize the glutathione peroxidase activity. Both a low glutathione peroxidase activity and a low glutathione reductase activity were found in pigmented tissue of frog liver when compared with unpigmented tissue of rat liver. In light of our results, we also report a hypothetical interrelationship between melanin and reduced glutathione: We believe that in pigmented cells the melanin could act as a reducing physiological agent replacing the glutathione in the reduction of hydrogen peroxide. This reducing action of melanin could cause a diminished need for GSH and therefore could provoke the low glutathione peroxidase and reductase activities in pigmented tissue.