Toxicity and mutual interactions of cadmium and zinc ions in normal and carcinogen-transformed mouse cells

The toxicity of chloride salts of physiological (zinc, manganese, nickel) and non physiological (cadmium) bivalent metal ions was studied in normal or carcinogen-transformed mouse embryo fibroblast cells. The dose response curves for toxicity to both types of cells exhibited similar shapes. The transformed cells, however, were about twice as sensitive to zinc toxicity as normal cells. When normal and transformed cells were grown together and incubated for several hours with an appropriate concentration of zinc, the malignant cells were selectively killed. Cadmium was much more toxic than the three other metal ions in both types of cells. Its toxic effect was reversed by simultaneous addition of zinc at nontoxic concentrations.Abbrevications CFA colony forming ability - MCA 3-methylcholanthrene     

Cerebroside and Sulfatide Biosynthesis in the Brain of Snell Dwarf Mouse: Effects of Thyroxine and Growth Hormone in the Early Postnatal Period

Snell dwarf mice (dw/dw) and normal mice (+/?) were injected with thyroxine (T₄) (1 μg/animal, four injections) and growth hormone (GH) (20 μg/animal, four injections) from the 5th to the 15th day of life. In the untreated dw/dw mouse brain, the specific activities of UDP-galactose:ceramide galactosyltransferase (CGalT), PAPS:cerebroside sulfotransferase (CST), and 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) were decreased by 28, 25, and 37%, respectively, compared with the control untreated +/? mice. The major effect of T₄ was an increase of the brain CNP in the +/? mice (+40%) and dw/dw mice (+111%). The treatment with T₄ also brought to normal the level of CGalT in dw/dw brain; a somewhat less marked effect on CST was observed. The treatment with GH had a great stimulatory effect on CNP: the specific activity of this enzyme increased by 40 and 69% in +/? and dw/dw mouse brain, respectively. On the contrary, no effect of GH on the CGalT activity was observe in this study. Our results suggest that T₄ and GH may have both independent and complementary actions on the myelin-associated enzymes during the early postnatal period of brain development.     

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S₁ of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.     

Mode de formation des inclusions lamellaires dans le poumon embryonnaire de poulet

Résumé Les pneumocytes granuleux, qui constituent l'un des principaux types cellulaires de l'épithélium pulmonaire, sont caractérisés par la présence de volumineuses inclusions osmiophiles lamellaires.Nous avons étudié l'apparition et l'origine de ces inclusions dans l'épithélium du poumon embryonnaire de Poulet, en l'examinant à différents stades du développement.Les premières inclusions lamellaires apparaissent dans le poumon de l'embryon de 16 jours. A ce stade, quelques lamelles concentriques entourent une zône centrale amorphe étendue; la périphérie des inclusions contient toujours de petites structures granulaires. Les jours suivants le nombre de cellules contenant des inclusions lamellaires augmente rapidement; en même temps, les lamelles deviennent plus nombreuses. A 19 jours, les inclusions lamellaires ont un aspect semblable à celui qu'elles ont dans les poumons d'animaux adultes.Dès l'apparition des ébauches pulmonaires, à 2 1/2 jours d'incubation, les cellules épithéliales contiennent des inclusions typiques: les inclusions granulaires. Ces organites sont caractérisés par un centre granulaire, qu'entouré un système membranaire. Ce système, simple chez le jeune embryon, évolue ensuite en se compliquant; chez l'embryon de 16 jours, il s'enroule en plusieurs couches autour de la masse centrale. Au moment où les premières inclusions lamellaires apparaissent, le nombre des inclusions granulaires augmente rapidement; on les trouve souvent étroitement associées à des vacuoles lipidiques.L'analyse des relations entre inclusions lamellaires, inclusions granulaires et vacuoles lipidiques suggère que l'inclusion lamellaire résulte de la collaboration entre une vacuole lipidique et plusieurs inclusions granulaires.

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Differentiation of lamellar inclusions in the chick embryonic lung

Summary The granular pneumocytes, one of the main cellular types of the lung epithelium, are characterized by the presence of large osmiophilic lamellar inclusions. The appearance and origin of these inclusions has been studied in the epithelium of chick embryonic lung at different developmental stages.Lamellar inclusions are first seen in the lung of 16 day old embryos. At this stage, few concentric lamellae surround a large amorphous center; the periphery of the inclusions always contains small granular structures. In the following days, the number of cells containing these lamellar inclusions increases rapidly, while their lamellae progressively become more numerous. In 19 day old embryos, the lamellar inclusions are similar to those in the lungs of adult animals.From the earliest formation of the bronchial primordia, their epithelial cells contain a number of typical granular inclusions. These organelles are characterized by a granular center, enclosed in a membranous system. This structure becomes more complex as the embryo develops; in the 16 day old embryo, the multilayered membranous system coils around the granular center. At the time when lamellar inclusions first appear, granular inclusions increase rapidly in number and are often found in close association with lipidic vacuoles.The relationships between lamellar inclusions, granular inclusions and lipidic vacuoles are discussed. The evidence suggests that a lamellar inclusion arises from the cooperation of several granular inclusions and a lipidic vacuole.

     

Failure of spermatozoa from T/t mice to fertilize in vitro is overcome by zona drilling

Failure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.     

Ultrastructural and histochemical changes in the murine zona pellucida during the final stages of oocyte maturation prior to ovulation

The ultrastructural morphology of the mouse zona pellucida was studied in preovulatory follicles from the ovaries of immature mice treated with exogenous gonadotrophins. The ovaries were fixed in the presence of cetylpyridinium chloride, which precipitates carbohydrates, so that their loss during fixation and processing is substantially reduced. The semi-thin araldite sections obtained from osmicated material were viewed by conventional light microscopy, while the ultra-thin sections were examined by transmission electron microscopy. A parallel series of semi-thin sections of non-osmicated ovaries was deresined and subsequently stained with periodic acid Schiff (PAS). The morphological appearance of the zona pellucida in preovulatory oocytes changed during the final stages of oocyte maturation. A close correlation was observed between the ultrastructural appearance of the zona pellucida and that observed following PAS staining during the period studied. Real differences were observed in the location, density, and distribution of glycoconjugates within the zona pellucida during the final stages of oocyte maturation prior to and immediately following germinal vesicle breakdown. Similar changes in the zona were observed in adult females autopsied during proestrus and oestrus. The changes in the density and distribution of glycoconjugates are likely to have important consequences for fertilization by affecting sperm-zona binding and sperm-egg interactions.     

Pregnant mares' serum gonadotropin source influences fertilization and fresh or thawed embryo development,but the effect is genotype specific

The influence of the source of pregnant mares' serum gonadotropin (PMSG) on the num ber, quality, and in vitro development of mouse embryos before and after freezing was evaluated among three genotypes: N:NIH(S), C57BL/6N, and C3H/HeN-MTV^?. Immature females were given PMSG from one of five commercial sources. Following col lection ( 116 hr later), embryos were evaluated for stage of development, and four-to eight-cell embryos were pooled within genotype and assigned to standardized fresh or freeze-thaw culture trials. Different PMSG sources stimulated the production of different num bers of total embryos (P < 0.05) but not necessarily more embryos suitable for freezing. Differences in embryo production among genotypes indicated that absolute embryo num bers using a single mouse genotype may not accurately reflect the potency of a specific gonadotropin source. The PMSG source also affected the ability of an embryo to survive in culture either immediately after collection or after frozen storage. The effect, however, was genotype specific, with some mouse strains being relatively insensitive to PMSG source, whereas gonadotropin source played a major role in determining in vitro viability in others. Development rates for freshly collected embryos differed, often inconsistently, from those of thawed embryos regardless of the PMSG source used, demonstrating that fresh embryo development cannot be used to estimate expected post-thaw survival. In vitro development of thawed embryos is influenced not only by genotype, but also the source of the gonadotropin used to promote follicular development and oocyte maturation. These findings may explain, in part, the wide variation in embryo viability and culture rates reported among laboratories and intraspecies animal populations.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

Direct effects of serotonin and ketanserin on the functional morphology of embryonic chick skin in vitro

Summary Different organotypical culture methods are used to test the direct effects of serotonin and ketanserin, a S₂, α₁, and H₁ receptor antagonist in vascular tissue, on fibroblasts and epidermal cells of embryonic chick skin in vitro. From light microscopic and electron microscopic analyses, we learn that serotonin enhances keratinization and differentiation, whereas ketanserin reduces differentiation in comparison to the control cultures. Incorporation data of fragments cultured with [³H]thymidine show that ketanserin, within a dose range from 0.05 to 5 μg/ml, stimulates proliferation. Serotonin at a concentration of 10 μg/ml slightly slows down proliferation, whereas lower doses of 0.1 and 1 μg/ml result in tritium activities that do not differ from control cultures. This investigation was financially supported by the National Fund of Scientific Research, Belgium, 3.0022.87.