Cytochemical and ultrastructural study of oocyte development of the crab Eriocheir sinensis. II. Oogenesis after removal of the eyestalk]

The effect produced by an eyestalk removal have been studied on Eriocheir females at different physiological stages. In juvenile and prepuberal crabs, the operation induces an important rise of the oocyte diameter. Only a few variations are observed in puberal females oocytes. Cytological changes are found at first at the nucleolar level. The granular area increases and the nucleolar vacuoles volume decreases. Then the granules (precursor material to endogenous yolk) disappear in the reticulum cisternae. At this time, the endogenous yolk seems essentially elaborated within yolk lobules. The envelope of these lobules is enhanced by ribosomes. In juvenile females (oocytes initially in previtellogenesis) exogenous yolk does not appear. Nevertheless in prepuberal females, following eyestalks deprivation, the oocytes, initially at the endogenous vitellogenesis stage, quickly reach the vitellogenesis second stage. In such oocytes, the microvilli development and pinocytose vesicles number are greater than normally. Cytochemical tests reactions do not demonstrate differences in the yolk material (endogenous and exogenous) nature from experimented oocytes and controls. In juvenile and prepuberal oocytes, the multivesicular bodies and lysosomes proliferation, the increase in glycogen and lipids amount express a metabolic disturbance resulting from an acceleration of growth processes. However in eyestalk-less prepuberal females no difference with the control oocytes was noticed.     

Evaluation of growth rate in adhering cell cultures using a simple colorimetric method

In this paper we present a simple colorimetric method for evaluating cell growth in adhering cell cultures. This technique is based on the staining of basophilic cellular compounds (mainly nucleic acids) with methylene blue. To check its reliability, we have compared the quantity of dye fixed by the cells with the number of cells released from the culture substratum by trypsinization, and with the total cellular protein content determined by the Lowry's method. In these experiments we have used human skin fibroblasts and human epithelial cells derived from the epithelium of the full-term umbilical cord. Like other alternative methods, the methylene blue colorimetric technique can be used in 96-well microplates and allows the rapid collection of large amounts of data. As an illustration, we report on the selection of optimal composition of culture medium for human skin fibroblasts.     

Culture of epithelial cell lines in chemically defined media on microcarriers in biogenerators

A rat liver epithelial cell line has been propagated on microcarriers in 11 or 21 laboratory culture vessels for cell culture in suspension on microcarriers (biogenerators) with Ham F10 or DME as basal synthetic culture medium either serum-supplemented (SSM), or serum-free (SFM), or serum- and protein-free (SPFM). Without serum, the use of DME allows a cellular growth in the biogenerator at least equivalent to that obtained in culture dishes. For the cultivation on microcarriers in SFM in a biogenerator the use during the first day of culture of spent serum-free medium previously incubated (SFMI) in confluent culture dishes avoids the substratum treatment with serum. Results concerning the Vero cell line cultured in SPFM are shown.     

Mode de formation des lamelles annelées résultant de l'évolution,en condition anhormonale,des ovocytes de Nereis diversicolor (Annélide Polychète)

Résumé L'évolution en l'absence d'hormone cérébrale provoque, dans les ovocytes de N. diversicolor, la formation de lamelles annelées cytoplasmiques (L.A.). Le processus débute par l'apparition de lames de réticulum à proximité du noyau (15 jours d'évolution à 15° C). Les lames se rapprochent ensuite du noyau et des pores apparaissent, situés le plus souvent face à ceux de l'enveloppe nucléaire (20ème jour). La différenciation des lames du réticulum en L.A. progresse ensuite depuis les éléments les plus proches du noyau vers les plus externes.Dans le cytoplasme des ovocytes ayant évolué à la température de 20° C, on assiste en outre à la formation d'empilements de citernes ergastoplasmiques. Ces citernes, non situées à proximité du noyau, subissent également une transformation progressive en L.A.L'étude cytochimique effectuée par digestion enzymatique a montré que la pepsine dégrade fortement le matériel dense des lamelles annelées. Ce matériel semble donc renfermer une teneur élevée en protéines auxquelles pourrait se trouver associé du RNA.

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Mode of formation of the annulate lamellae of oocytes of Nereis diversicolor developing in the absence of brain hormone

Summary Experimental elimination of brain activity leads to the formation of cytoplasmic annulate lamellae (L.A.) in oocytes of N. diversicolor. This process begins with the appearance of cisternae of endoplasmic reticulum in the vicinity of the nucleus (15 days of development at 15° C). Then, the cisternae move closer to the nucleus and pores appear, often facing the pores of the nuclear envelope (20th day). The differentiation of endoplasmic reticulum into L.A. progresses from the elements closest to the nucleus to the more peripheral ones.In the cytoplasm of oocytes which developed at 20° C, stacks of cisternae of rough endoplasmic reticulum have been observed. These cisternae, which are not localized near the nucleus, also undergo a progressive transformation into annulate lamellae.A cytochemical study, based on enzymatic digestion, shows that pepsin strongly digests the dense material of L.A. This material, therefore, seems to contain a high amount of proteins, perhaps associated with RNA.

     

Oxygen consumption during septic shock. Effects of inotropic drugs

Septic shock may be defined as a clinical entity wherein a patient has an inadequate peripheral metabolism in the presence of circulating bacteria. The demands for metabolic requirements of the tissues and hence for oxygen transport to these tissues are then markedly high. The average response to increased metabolism in such patients is a percentage increase in cardiac output that is comparable to the percentage increase in oxygen consumption while oxygen extraction rate does not change or even decreases in severe or/and advanced septic shock. This emphasizes the high priority placed by the body on the ability to increase blood flow from the heart in the presence of increased metabolic demands due to sepsis. Concerning the myocardium, oxygen consumption is low in hyper- and hypodynamic states of septic shock, probably and partially due to marked arterial vasodilatation. However, in hypodynamic states, the lower value of perfusion pressure may account for a decrease in myocardial oxygen supply, especially in subendocardial areas and may be responsible for myocardial ischaemia, more especially as myocardial oxygen extraction as well as systemic oxygen extraction is impaired. The goal of therapeutics is to improve oxygen availability through: (1) maintenance of haemoglobin levels and (2) increases in stroke volume using inotropic drugs since these drugs may produce a rise in myocardial oxygen supply higher than their drug-induced increase in oxygen requirements in hypodynamic states of septic shock.     

Etude cytochimique et ultrastructurale de l'évolution ovocytaire de Nereis pelagica L. (Annélide Polychète)

Résumé Les ovocytes de N. pelagica d'un diamètre inférieur à 100 ne renferment dans leur cytoplasme que des inclusions lipidiques et vitellines. Les lobules vitellins s'accroissent par l'adjonction de vésicules golgiennes.L'approche de la maturité sexuelle est caractérisée par l'apparition de mucopolysaccharides acides. Corrélativement à l'élaboration de ce matériel, l'appareil de Golgi présente, à ce stade, de profondes modifications morphologiques: dilatation des saccules distaux et libération dans le cytoplasme de vacuoles golgiennes. Les processus de vitellogenèse cependant ne sont pas entièrement stoppés.Dans les ovocytes matures, les lobules mucopolysaccharidiques forment une gangue corticale. Les dictyosomes sont refoulés vers l'intérieur du cytoplasme et présentent des figures d'involution.La présence de lamelles annelées intra-nucléaires a été observée de façon constante dans tous les ovocytes matures examinés.

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Cytochemical and ultrastructural study of oocyte development in Nereis pelagica L.I. Normal ovogenesis

Summary The oocytes of N. pelagica, the diameter of which is smaller than 100 , contains only lipid and yolk inclusions in their cytoplasm. The yolk bodies grow by confluence of Golgi vesicles.The advent of the sexual maturity is marked by the appearance of acid mucopolysaccharides. Concomitantly with the production of this material, the Golgi apparatus shows typical morphological modifications: dilatation of the distal saccules and release of Golgi vacuoles into the cytoplasm. However vitellogenesis is not completely terminated at this point.In the mature oocytes the mucopolysaccharid material forms a cortical layer. The dictyosomes are pushed toward the center of the cytoplasm and show signs of degeneration.The presence of intranuclear annulate lamellae is a constant feature in all mature oocytes examined.

     

Biosynthesis of metabolites of 2-acetylaminofluorene by isolated hepatocytes of rats: study by gas chromatography-mass spectrometry

A new technique for the derivatization, the separation and the quantification of 2-acetylaminofluorene (2-AAF) and its metabolites biosynthetized by freshly isolated hepatocytes was developed combining gas chromatography and mass spectrometry. Analysis of the different metabolites was carried out after their derivatization into tertbutyldimethylsilyl compounds. Freshly isolated hepatocytes metabolized 2-AAF and produced five aryl-hydroxylated compounds as well as the N-hydroxy-2AAF and the 2-aminofluorene. The metabolites were found under their free and conjugated forms.     

Continuous production of large amounts of monoclonal immunoglobulins in hollow fibers using protein-free medium

The performance of a protein-free medium was compared in culture flasks with a serum-supplemented medium and with a serum free medium in terms of cell growth and monoclonal antibody production by a murine hybridoma. We present results of continuous production in hollow fiber culture systems using serum-free medium and protein-free medium. In protein-free medium, it has been possible to produce large quantities of monoclonal antibody with a productivity similar to that obtained in serum-free medium. After a two steps purification process, monoclonal antibodies were characterized by SDS-PAGE, High Performance Size Exclusion Chromatography and Free Solution Capillary Electrophoresis. SDS-PAGE and high performance chromatography analysis have showed that purified monoclonal antibodies produced in serum-free medium or protein-free medium were similar. Furthermore, Capillary Electrophoresis characterization revealed that both MAbs were constituted by three isoforms with equivalent electrophoretic mobilities.Abbreviations CHES 2-(N-Cyclohexylamino)ethane-sulfonic acid - ECS Extracapillary Space - FSCE Free Solution Capillary Electrophoresis - HPSEC High Performance Size Exclusion Chromatography - ICS Intracapillary Space - MAb Monoclonal Antibody - PFM Protein-Free Medium - SFM Serum-Free Medium - SSM Serum-Supplemented Medium