[Relation between metabolism of trace elements and the antioxidant defense system in chronic nephropathies]. / Vztah medzi metabolizmom stopových prvkov a obranným antioxidacným systémom pri chronických nefropatiách.

BACKGROUND: Trace elements--zinc, copper and selenium are part of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GHPx). In renal diseases changes in the trace element metabolism may influence the equilibrium in the antioxidant defence system and enhance the toxic effect of reactive oxygen molecules. METHODS AND RESULTS: The authors examined in 53 children with chronic neprophathies (34 with chronic glomerulonephritis, 11 with chronic renal failure and 8 children included in a chronic dialyzation programme) zinc and selenium in blood and urine and antioxidant enzymes SOD and GHPx in red blood cells. The lowest SOD activity (737 +/- 219 U/g Hb, p < 0.01) and serum zinc concentration (12.9 +/- 3.2, p < 0.05) were found in children in the terminal stage of uraemia, GHPx was as compared with the group of healthy children elevated in all groups of sick children. In dialysed children GHPx was highest (p < 0.01) and correlated with urinary selenium concentrations (r = -0.86, p < 0.05). The SOD activity depended on the serum copper concentrations (r = 0.78, p < 0.05). The highest renal zinc and selenium excretion was recorded in patients with glomerulonephritis (p < 0.05). CONCLUSIONS: The results support the hypothesis on a causal relationship between trace elements and antioxidant enzymes.

Chemically enucleated mouse oocytes: ultrastructure and kinetics of histone H1 kinase activity.

The objective of the study was to characterize the ultrastructure changes and biochemical mechanisms underlying the expulsion of the entire chromosome complement in chemically enucleated mouse oocytes. The ultrastructural studies demonstrated that the morphology of cytoplasts produced by etoposide-cycloheximide treatment were indistinguishable from intact metaphase I and II oocytes. Moreover, polar bodies formed by chemical enucleation were in almost all cases completely separated from the parent cytoplast and differed from normal polar bodies only in their chromatin content morphology and because they contained a slightly higher number of cytoplasmic organelles. The mode of polar body formation, however, in normal and chemically enucleated oocytes differs substantially: spindle involvement is important for normal polar body extrusion but plays no part in the protracted expulsion of chromosomes during chemical enucleation. After etoposide-cycloheximide treatment, histone H1 kinase activity remains high for the ensuing 6-8 h before declining gradually to basal levels 14 h after treatment. The expulsion of the polar body occurred only after the slowly declining H1 kinase activity reached basal levels. The activity of this kinase rose sharply to reach maximal levels within 4 h when the enucleated oocytes were removed from the inhibitor-supplemented medium and placed in normal medium. The findings in this paper indicate that cytoplasts produced by chemical enucleation are morphologically normal, thus suggesting that these enucleated cells are suitable for cloning studies. Although effective in mouse oocytes, we postulate that certain modifications to the enucleation technology are necessary before a reliable non-invasive protocol for ungulate oocytes will be available.

Oocyte-specific modulation of female pronuclear development in mice.

Cell fusion experiments were undertaken to determine both whether oocytes possess, in a stage-specific manner, suppressors of pronuclear development and whether these factors could be used to modulate female pronuclear development. The fusion of telophase II eggs obtained 2 hr after activation (A2 eggs) to germinal vesicle (GV)-staged oocytes (GV x A2) suppresses female pronuclear development. The cytoplasm of GV x A2 heterokaryons contains, at 10 hr postfusion, a micropronucleus and a GV which is significantly larger (approximately x1.6) than normal. Fusion of early pronucleate eggs (3 hr postactivation, A3) to GV-staged oocytes (GV x A3) results in the formation of a retarded half-sized pronucleus and a slightly enlarged (approximately x1.2) GV at 10 hr postactivation. Normal pronuclear formation occurs when eggs at 4 hr postactivation (A4) are fused to GV oocytes (GV x A4). Although pronuclear size is suppressed in heterokaryons formed when GV-staged oocytes are fused to eggs activated 2-3 hr earlier (A2 or A3 stage), entry into S-phase and DNA synthesis is not inhibited even in the micropronuclei. Meiotic progression in the oocyte germinal vesicle is arrested after fusion to activated eggs throughout the period during which the pronucleus is undergoing G1 and S-phase. However, at the end of S-phase in the pronucleate partner, germinal vesicle breakdown occurs, cell cycle progression is accelerated, and both nuclei reach M-phase by 12 hr postactivation. That suppressors of pronuclear development are found only in GV (G2)-staged oocytes, and not in mitotic cells at the same cell cycle stage, was demonstrated by fusing G2-staged blastomeres to A2-staged eggs (G2 mitotic x A2). By contrast to the micropronuclei formed in GV x A2 heterokaryons, normal pronuclear development occurred in G2 mitotic x A2 heterokaryons. Our results show that suppressor activity, present only in GV oocytes, restricts female pronuclear development, but only in the first 3 hr after activation. We propose to use the ability to modulate either female (this study) or male pronuclear formation in studies (i) on imprinting and (ii) on the developmental consequences of early asynchrony between male and female pronuclear development.

Nuclear transplantation in pigs: M-phase karyoplast to M-phase cytoplast fusion.

Porcine embryonic fibroblasts in M-phase were isolated from confluent cultures by selective detachment and fused to homologous metaphase II enucleated oocytes. The fusion products were analysed by light and electron microscopy (EM). The fusion treatment, per se, did not induce the disappearance of the maturation promoting factor (MPF) in the cloned embryos and the transferred chromatin remained condensed for up to 10 h. Spontaneous decondensation started after this time and, at about 20 h post-induction of fusion, a single large nucleus was present in the cytoplasm. The prolonged exposure of introduced chromatin to MPF resulted in a total morphological remodelling as assessed by the nucleolar morphology observed by EM in all the specimens.

Response of avian nuclei to mammalian maturation promoting factor (MPF).

Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.

Cytoplasmic argyrophilic protein(s) in cumulus-oocyte complexes: a preliminary study.

The presence of acidic argyrophilic protein(s) in cumulus cells and oocytes was examined in bovine, ovine, porcine and murine cumulus-oocyte complexes (COCs) using a specific silver-staining method. The COCs were isolated from small (1 mm) or large (3-5 mm) antral follicles; they were fixed immediately after slaughter (first group) or after transfer to the laboratory (ca 60 min, second group) and then silver stained according to Likovskí and Smetana (1981). The argyrophilic proteins were accumulated mainly in foot processes of cumulus cells on oocytes isolated from large bovine follicles. The intensity of the reaction was less evident in small follicles. The intensity of staining gradually disappeared from cumulus cells as the maturation continued. Moreover, we were unable to detect similar strong labelling in mouse, pig and sheep COCs. Such a chronology suggests that argyrophilic protein(s) may play a role in the transition from meiotic arrest to resumption in bovine oocytes. Our results also suggest that preliminary events of maturation may occur just after slaughter, ie a relatively long time before the COCs are isolated and cultured in vitro.

Localization of 2-glucuronidase in the marginal sinus of rat spleen.

The study demonstrated lysosomal 2-glucuronidase activity localized in the marginal sinus epithelium of rat spleen. Immunologically, the marginal sinus is a key structure of the spleen and its enzymatic apparatus differs from that of the cells of the red pulp venous sinuses. The possible physiological significance of this finding is briefly discussed.