Ultrastructure and intracellular calcium response during activation in vitrified and slow-frozen human oocytes.

BACKGROUND: The sensitivity of human oocytes to cryodamage may compromise their developmental competence following cryopreservation. Herein, we compared the ultrastructure and the response to the calcium (Ca²âº) ionophore A23187 of fresh, slow-frozen and vitrified metaphase II (MII) human oocytes. METHODS: Supernumerary fresh MII oocytes, donated under written informed consent, were cryopreserved through either a slow cooling procedure based on propane-1,2-diol and 0.3 M sucrose or a closed vitrification system based on dimethylsulphoxide (DMSO) and ethylene glycol (EG). Ultrastructure of fresh and cryopreserved oocytes was assessed by transmission electron microscopy and compared through morphometrical analysis; intracellular calcium ([Ca²âº](i)) dynamics was studied by evaluating the response to the Ca²âº ionophore A23187. RESULTS: Morphometric analysis demonstrated a markedly higher proportion of oocytes with large vacuoles, inward displacement of organelles from the pericortical toward the deep cytoplasm, and mitochondrial damage in slow-frozen compared with both fresh and vitrified oocytes. A23187 increased the [Ca²âº](i) in all oocyte groups and the peak average increase in slow-frozen oocytes was significantly higher than in both fresh and vitrified oocytes. Moreover, the ability of slow-frozen oocytes to recover [Ca²âº](i) to basal levels was significantly reduced compared with both fresh and vitrified oocytes. CONCLUSIONS: Closed vitrification based on DMSO and EG preserves the ultrastructural features and the ability to respond to the Ca²âº ionophore A23187 significantly better than does slow freezing with 0.3 M sucrose. Damage to organelles involved in the [Ca²âº](i) modulation might reduce the developmental competence of cryopreserved oocytes.

Ultrastructure of human mature oocytes after slow cooling cryopreservation with ethylene glycol.

The morphological characteristics of frozen-thawed human mature oocytes (n = 12) were studied by light and transmission electron microscopy following cryopreservation using a slow cooling protocol including increasing concentrations of ethylene glycol (0.5-1.5 mol/l) and sucrose 0.2 mol/l in the freezing solution. Fresh human mature oocytes (n = 12) were used as controls. Fresh and frozen-thawed oocytes appeared rounded in section, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Disorganization of mitochondria-smooth endoplasmic reticulum aggregates and a decreased complement of microvilli and cortical granules were frequently observable in frozen-thawed oocytes. Increased density of the inner zona pellucida, possibly related to the occurrence of zona 'hardening', was sometimes found associated with a reduced amount of cortical granules. In addition, delamination of the zona pellucida was evident in some frozen-thawed samples. Finally, numerous vacuoles and secondary lysosomes were detected in the ooplasm of most frozen-thawed oocytes. In conclusion, frozen-thawed oocytes treated with ethylene glycol may show a variety of ultrastructural alterations, possibly related, at least in part, to the use of this cryoprotectant. Thus, the ethylene glycol-based protocol of slow cooling herein described does not seem to offer significant advantages in terms of oocyte structural preservation.

Wastewater toxicity of tannin- versus chromium-based leather tanneries in Marrakesh, Morocco.

The toxicity of leather tanning wastewater from a traditional tannery (TT), which is based on vegetable tannin (VT), was compared with wastewater from a tannery combining the use of chromium-based tanning (CT) with VT-based tanning operations. Wastewater samples from a TT and a CT plant as well as from five sewer sampling points were collected in Marrakesh, Morocco, and the concentrations of VT and some selected inorganics were measured. A set of bioassays were used to test wastewater toxicity in sea urchin (Paracentrotus lividus) embryos and sperm, in Daphnia magna, and in marine microalgae (Dunaliella tertiolecta). Toxicity end points included: (1) developmental defects, embryonic mortality, sperm fertilization success, and offspring damage in sea urchins; (2) D. magna immobilization; and (3) algal growth rate inhibition. Toxicity tests on TT and CT effluents (TTE and CTE) were run at dilutions ranging from 0.1% to 2% (sea urchins and algae) or up to 12% in D. magna. Parallel bioassays were run on VT extract (VTE) at nominal tannin concentrations ranging from 0.1 to 10 mg l(-1). The results showed higher toxicity of CTE compared with TTE. CTE toxicity in sea urchins and algae showed concentration-related trends, whereas TTE exerted hormetic effects at levels of 0.1% to 0.2% and toxic effects at levels >or=1%. The same trends were observed for VTE, suggesting a prevailing role of tannin in TTE-associated effects. The moderate wastewater toxicity of VT-based tanneries might prompt interest in the VT tanning process.

Permeability of human oocytes to ethylene glycol and their survival and spindle configurations after slow cooling cryopreservation.

BACKGROUND: To develop novel cryopreservation methods, we estimated the permeability coefficients Lp (hydraulic conductivity) and P(EG) (cryoprotectant permeability) of mature human oocytes after exposure to ethylene glycol (EG) and tested the efficiency of a multi-step slow cooling protocol based on this cryoprotectant. METHODS: Oocytes were perfused with 1.5 mol/l EG for 10 min. Oocyte volume at each time point was calculated and normalized to the original volume. Slow cooling was conducted by exposing oocytes to increasing EG concentrations (0.5, 1.0 and 1.5 mol/l n = 155) or 1.5 mol/l of propane-1,2-diol (PrOH) n = 102. Oocytes which survived cryopreservation n = 80 and fresh oocytes n = 73 were prepared for confocal microscopy analysis of the meiotic spindle. RESULTS: During EG exposure, oocytes underwent an abrupt 50% volume reduction. Complete recovery of the initial volume was not observed. From the values of a best fit plot, the coefficients Lp = 0.82 +/- 0.29 microm min(-1) atm(-1) (mean +/- SD) and P(EG) 0.10 +/- 0.01 microm s(-1) were generated. Survival rates after freezing with EG were lower than with PrOH (51.6 versus 71.5%, respectively, P < 0.05). The frequencies of normal spindle configuration were lower in frozen EG and frozen PrOH oocytes compared with fresh oocytes (53.8, 50.9 and 66.7%, respectively, P < 0.05). CONCLUSIONS: The oocyte plasmalemma possesses limited permeability to EG and EG exposure causes considerable osmotic stress. However, post-thaw rates of survival and normal meiotic spindle organization may be preserved by protocols which are designed in order to minimize osmotic stress.

Hormetic versus toxic effects of vegetable tannin in a multitest study.

Tannin from mimosa trees (Acacia sp.) utilized in traditional leather tanning was tested for toxicity in sea urchin (Sphaerechinus granularis and Paracentrotus lividus) embryos and sperm, marine, and freshwater algae (Selenastrum capricornutum and Dunaliella tertiolecta), and Daphnia magna. Based on a two-step tanning procedure used in traditional tanneries, two mimosa tannin preparations, i.e., fresh tannin (FT) and used tannin (UT), were tested as suspensions. The early results in S. granularis embryos showed that UT exerted lower acute toxicity than FT, namely, 1 vs 100 mg/L, to obtain 100% mortality, respectively. Subsequent bioassays were conducted on fresh tannin water extracts (TWE) corresponding to nominal tannin concentrations ranging from 0.1 to 30 mg/L. Developmental toxicity, up to embryonic mortality was exerted by TWE at levels > 1 mg/L, S. granularis being more sensitive than P. lividus embryos/larvae. At the concentration of 0.1 mg/L, the frequencies of larval malformations were significantly lower than in controls. This positive stimulatory effect (currently termed as hormesis) was observed in extended numbers of culture replicates (up to 14) and was significant in the embryo cultures characterized by a relatively poor control quality (with < 70% viable larvae in controls), whereas this effect was not observed in good-quality cultures (with > or = 70% viable larvae in controls). Cytogenetic analysis of S. granularis embryos reared in FT or UT suspensions (1 mg/L to 1 g/L) showed mitotoxic effects (decrease in active mitoses per embryo) in FT-exposed, but not in UT-exposed embryos. Mitotic aberrations were significantly increased by 10 mg/L UT. Sperm fertilization success in both sea urchin species showed an increasing fertilization rate (FR) up to 0.3 mg/L TWE and a dose-related decrease in FR up to 30 mg/L. Again, the offspring of P. lividus sperm exposed to TWE (0.1 and 0.3 mg/L) showed a decrease in larval malformations compared to controls, whereas a dose-related increase in developmental defects was observed in the offspring of P. lividus sperm exposed to higher TWE levels (1 to 30 mg/L). Algal cell growth bioassays in two species (S. capricornutum and D. tertiolecta) also showed a maximum growth at TWE levels ranging from 0.3 to 3 mg/L and a subsequent decline up to 30 mg/L TWE. D. magna bioassays resulted in daphnid immobilization by TWE concentrations ranging from 100 to 300 mg/L. The results demonstrate that tannins utilized in traditional leather tanning industry may raise concern of environmental damage at relatively high concentrations, whereas low-level tannins may result in hormetic effects. The present study also points to the need for bioassay design that should rely on adequate criteria in control quality, allowing to detect both inhibitory and hormetic effects.

Factors affecting R6 fungicide toxicity on sea urchin fertilization and early development: roles of exposure routes and mixture components.

A technical fungicide mixture, R6 and its components, cymoxanil (CYM) and cupric oxychloride (Cu-OCl), were tested by sea urchin bioassays (Paracentrotus lividus and Sphaerechinus granularis). A set of toxicity endpoints was evaluated including both lethal and sublethal effects with the following endpoints: (a) acute embryotoxicity, (b) developmental defects, (c) changes in sperm fertilization success, (d) transmissible damage from sperm to the offspring, and (e) cytogenetic abnormalities. Acute effects on developing embryos were observed as early (prehatch) mortality at R6 levels > or =25 microg/ml. The pesticide mixture R6 was tested at realistic concentrations, ranging from 25 ng/ ml to 2.5 microg/ml, and the two components, CYM and Cu-OCl, were tested, either alone or in mixture, at concentrations equal to their levels in the corresponding R6 solutions. R6 was either dissolved in filtered seawater (water only, W-O), or spiked in "pristine" silt-clay sediment or soil samples before bioassays. Developmental toxicity of R6, following W-O dissolution, displayed a significant dose-related increase of larval malformations and differentiation arrest at concentrations of 750 ng/ml to 2.5 microg/ml both in P. lividus and in S. granularis larvae. Developmental toxicity was removed in spiked sediment up to R6 nominal levels (25 microg/ml), 10-fold above the embryotoxic R6 levels in W-O exposure. No significant developmental toxicity was exerted by CYM or Cu-OCl (W-O exposure) up to their concentrations equivalent to 2.5 microg/ml R6. The laboratory-prepared mixture of CYM and Cu-OCl, in the same concentration range, only resulted in minor effects, as larval retardation, suggesting the presence of toxic impurities (or additional components) in the R6 formulation. When sperm from either P. lividus or S. granularis were exposed to R6 before fertilization, a W-O exposure resulted in a dose-related decrease in fertilization of P. lividus sperm (up to 250 microg/ml R6), whereas S. granularis sperm underwent a significant increase of fertilization rate at the highest R6 nominal levels (up to 25 microg/ml). Equivalent CYM or Cu-OCl levels were ineffective on sperm fertilization success in both species. The offspring of S. granularis sperm exposed to 25 microg/ml R6 showed a significant increase in larval malformations, which were not detected in the offspring of R6-exposed P. lividus sperm. Again, CYM or Cu-OCl was unable to exert any transmissible damage from sperm to the offspring in either species. The present study raises the case of possible discrepancies between toxicity of a technical mixture and of its analytical-grade components, also providing evidence for a loss of pesticide toxicity following dispersion in an environmental matrix such as sediment or soil.

The Rhizobium GstI protein reduces the NH4+ assimilation capacity of Rhizobium leguminosarum.

We show that the protein encoded by the glutamine synthetase translational inhibitor (gstI) gene reduces the NH4+ assimilation capacity of Rhizobium leguminosarum. In this organism, gstI expression is regulated by the ntr system, including the PII protein, as a function of the nitrogen (N) status of the cells. The GstI protein, when expressed from an inducible promoter, inhibits glutamine synthetase II (glnII) expression under all N conditions tested. The induction of gstI affects the growth of a glutamine synthetase I (glnA-) strain and a single amino acid substitution (W48D) results in the complete loss of GstI function. During symbiosis, gstI is expressed in young differentiating symbiosomes (SBs) but not in differentiated N2-fixing SBs. In young SBs, the PII protein modulates the transcription of NtrC-regulated genes such as gstI and glnII. The evidence presented herein strengthens the idea that the endocytosis of bacteria inside the cytoplasm of the host cells is a key step in the regulation of NH4+ metabolism.

The role of oxidative stress in developmental and reproductive toxicity of tamoxifen.

The antiestrogen tamoxifen (TAM) is widely used as a drug against breast cancer and is currently being tested as a chemopreventive agent. However, a number of studies showed genotoxic and carcinogenic effects of TAM. These effects are thought to be related to oxygen radical overproduction which occurs during TAM metabolic activation. There is no evidence, thus far, on TAM toxicity to embryos and gametes. The present study was designed to elucidate the mechanisms of TAM-induced developmental, reproductive and cytogenetic toxicity towards sea urchin (SU) embryos with regard to the possibility of TAM-initiated oxidative stress. Embryo cultures from SU were subjected to long-term (throughout embryogenesis) or short-term (two hours) incubation with TAM at concentrations from 10(-8) to 10(-5) M. The experiments on TAM-induced toxicity to gametes were carried out with SU sperm, or unfertilized eggs, suspended in TAM (10(-8) to 10(-6) M). To assess the effects of TAM to embryos or to gametes, developmental defects, embryonic mortality, fertilization success, and cytogenetic abnormalities were scored. Oxidative damage to DNA and lipids was detected by measurements of 8OHdG levels and lipid peroxidation, respectively. Reactive oxygen species (ROS) production by eggs and embryos was recorded by luminol-dependent chemiluminescence (LDCL) and cytochrome c reduction methods. The changes in activities of SU superoxide dismutase (SOD) and catalase were also evaluated. TAM exerted: a) early embryonic mortality to exposed embryos and to the offspring of exposed eggs; b) developmental defects to the offspring of exposed sperm; c) decrease in sperm fertilization success, and d) cytogenetic effects in the offspring of exposed sperm or eggs. These morphological effects corresponded to the state of oxidative stress in SU embryos (increased oxidative damage to DNA and lipids and induction of antioxidant enzymes). Since TAM did increase significantly ROS production by embryos, it is suggested that TAM may be metabolically activated by SU embryonic oxidases and peroxidases, which in turn could be induced by TAM. The present study provides further support to the utilization of the SU system as a useful model to help elucidate mechanisms of chemical teratogenesis and carcinogenesis.

Diepoxybutane and mitomycin C toxicity is associated with the induction of oxidative DNA damage in sea urchin embryos.

Diepoxybutane (DEB)- and mitomycin C (MMC)-associated toxicity was investigated in embryos from the sea urchin (SU) species Sphaerechinus granularis. DEB- and MMC-induced toxicity resulted in S. granularis embryos and larvae at concentrations ranging 10(-5) to 10(-4) M DEB, and 3 x 10(-6) to 3 x 10(-5) M MMC, in terms of larval malformations, developmental arrest and mortality. The formation of DNA oxidative damage, 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured in DEB- and in MMC-exposed embryos (at gastrula stage). A dose-dependent increase in 8-OHdG levels was observed that was significantly correlated with DEB- and MMC-induced developmental defects. The results lend further support to the body of evidence associating both DEB and MMC toxicity with oxidative stress, including DNA oxidative damage.

Inhibition of glutamine synthetase II expression by the product of the gstI gene.

We report the identification of a previously unrecognized gene that is involved in the regulation of the Rhizobium leguminosarum glnII (glutamine synthetase II) gene. This gene, which is situated immediately upstream of glnII, was identified by means of a deletion/complementation analysis performed in the heterologous background of Klebsiella pneumoniae. It has been designated gstI (glutamine synthetase translational Inhibitor) because, when a complete version of gstI is present, it is possible to detect glnII-specific mRNA, but neither GSII activity nor GSII protein. The gstI gene encodes a small (63 amino acids) protein, which acts in cis or in trans with respect to glnII and is transcribed divergently with respect to glnII from a promoter that was found to be strongly repressed by the nitrogen transcriptional regulator NtrC. A mutated version of GstI lacking the last 14 amino acids completely lost its capacity to repress glnII expression. Our results indicate that gstI mediates the translation inhibition of glnII mRNA and, based on in silico analyses, a mechanism for GstI action is proposed.

Redox-dependent toxicity of diepoxybutane and mitomycin C in sea urchin embryogenesis.

The effects and mechanisms of action of diepoxybutane (DEB) and mitomycin C (MMC) were investigated on sea urchin embryogenesis, (Sphaerechinus granularis and Paracentrotus lividus). DEB- and MMC-induced toxicity was evaluated by means of selected end-points, including developmental defects, cytogenetic abnormalities and alterations in the redox status [oxygen-dependent toxicity, Mn-superoxide dismutase (MnSOD) and catalase activities and glutathione (GSH) levels]. Both DEB and MMC exhibited developmental toxicity (at concentrations ranging from 3 x 10(-5) to 3 x 10(-4) M and 3 x 10(-6) to 3 x 10(-5) M, respectively) expressed as larval abnormalities, developmental arrest and mortality. The developmental effects of both compounds were significantly affected by oxygen at levels ranging from 5 to 40%. These results confirmed previous evidence for oxygen-dependent MMC toxicity and are the first report of oxygen dependence for DEB toxicity. Both DEB and MMC exerted significant cytogenetic abnormalities, including mitotoxicity and mitotic aberrations, but with different trends between the two chemicals, at the same concentrations as exerted developmental toxicity. The formation of reactive oxygen species was evaluated using: (i) luminol-dependent chemiluminescence (LDCL); (ii) reactions of the main antioxidant systems, such as GSH content and MnSOD and catalase activities. The results point to clear-cut differences in the effects induced by DEB and MMC. Thus, DEB suppressed GSH content within the concentration range 10(-7)-3 x 10(-5) M. The activity of catalase was stimulated at lower DEB levels (10(-7)-10(-6) M) and then decreased at higher DEB concentrations (> or =10(-5) M). Increasing MMC concentrations induced LDCL and MnSOD activity (> or =10(-6) M) greatly and modulated catalase activity (10(-7) - 10(-6) M). GSH levels were unaffected by MMC. The results suggest that oxidative stress contributes to the developmental and genotoxic effects of both toxins studied, although through different mechanisms.

The soybean ENOD40(2) promoter is active in Arabidopsis thaliana and is temporally and spatially regulated.

The ENOD40 gene is induced early during Rhizobium-legume symbiosis and has probably a primary role in the nodule organogenesis. In this paper we show that the 1.7 kb 5'-flanking region of the GmENOD40(2) is able to drive the expression of a gusA-int marker in transgenic Arabidopsis thaliana. The promoter activity is developmentally regulated and the major activity is detected in the root and in the stigma.

The Rhizobium etli metZ gene is essential for methionine biosynthesis and nodulation of Phaseolus vulgaris.

A mutant strain (CTNUX23) of Rhizobium etli carrying Tn5 unable to grow with sulfate as the sole sulfur source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a metZ (O-succinylhomoserine sulfhydrylase)-homologous gene. The CTNUX23 mutant strain had a growth dependency for methionine, although cystathionine or homocysteine, but not homoserine or O-succinylhomoserine, allowed growth of the mutant. RNase protection assays showed that the metZ-like gene had a basal level of expression in methionine- or cysteine-grown cells, which was induced when sulfate or thiosulfate was used. The metZ gene was cloned from the parent wild-type strain, CE3, and the resulting plasmid pAR204 relieved, after transformation, the methionine auxotrophy of both strains CTNUX23 of R. etli and PAO503(metZ) of Pseudomonas aeruginosa. Unlike strain CE3 or CTNUX23 (pAR204), strain CTNUX23 showed undetectable levels of O-succinylhomoserine sulfhydrylase activity. Strain CTNUX23 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) or to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris, unless methionine was added to the growth medium. These data and our previous results support the notion that cysteine or glutathione, but not methionine, is supplied by the root cells to bacteria growing inside the plant.

Emerging diseases: a global threat.

A growing and globalizing threat of emerging and re-emerging diseases is best addressed through reliance on rapid detection, diagnosis, and containment. The efficiency and success of this approach depends on intricate networking and collaboration among all stakeholders including intergovernmental and nongovernmental organizations and specialized agencies in the developed and developing countries. Such cooperation, while focusing on eliminating a growing threat, also helps avoid duplication of effort and improves use of scarce financial resources. This review provides a summary of the problem of emerging/re-emerging diseases and the effort being directed at controlling the threat. Opportunities are identified for a more coordinated approach to addressing the problem.

A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation.

By its inability to grow on sulfate as the sole sulfur source, a mutant strain (CTNUX8) of Rhizobium etli carrying Tn5 was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a cysG (siroheme synthetase)-homologous gene. By RNase protection assays, it was established that the cysG-like gene had a basal level of expression in thiosulfate- or cysteine-grown cells, which was induced when sulfate or methionine was used. Unlike its wild-type parent (strain CE3), the mutant strain, CTNUX8, was also unable to grow on nitrate as the sole nitrogen source and was unable to induce a high level of nitrite reductase. Despite its pleiotropic phenotype, strain CTNUX8 was able to induce pink, effective (N2-fixing) nodules on the roots of Phaseolus vulgaris plants. However, mixed inoculation experiments showed that strain CTNUX8 is significantly different from the wild type in its ability to nodulate. Our data support the notion that sulfate (or sulfite) is the sulfur source of R. etli in the rhizosphere, while cysteine, methionine, or glutathione is supplied by the root cells to bacteria growing inside the plant.

L-methionine induces stage-dependent changes of differentiation and oxidative activity in sea urchin embryogenesis.

This study was to investigate developmental toxicity of some selected low molecular weight antioxidants, by utilising sea urchin embryos and gametes as model system. Sea urchin embryos or sperm were exposed at different developmental stages to L-methionine or some selected low molecular weight antioxidants: a) N-acetylcysteine; b) L-carnosine; c) L-homocarnosine, and d) L-anserine. L-methionine displayed developmental toxicity at levels > or = 10(-5) M, whereas the other agents tested were mostly active at levels > or = 10(-4) M. When embryos were exposed to 10(-4) M L-methionine or N-acetylcysteine at different developmental stages, the most severe effects were exerted by early exposures (0 to 2 hr after fertilisation), whereas later exposures turned to lesser or no effects. Cytogenetic analysis of L-methionine-exposed embryos showed a significant mitogenic effect and increase of mitotic aberrations. Fertilisation success was decreased by L-methionine (10(-6) M to 10(-3) M) added at the moment of fertilisation, with increasing developmental and cytogenetic abnormalities in the offspring. The formation of reactive oxygen species in embryos and gametes was determined by: a) analysing the DNA oxidative product, 8-hydroxy-2'-deoxyguanosine (8-OHdG), and b) luminol-dependent chemiluminescence. The results showed that: 1) 8-OHdG levels were increased during embryogenesis; 2) fertilisation was associated with a double-wave luminol-dependent chemiluminescence emission; 3) luminol-dependent chemiluminescence was maximal in cleavage, declining down to zero in plutei, and 4) an embryotoxic L-methionine or N-acetylcysteine level (10(-4) M) turned to a decrease in reactive oxygen species formation. The data suggest that L-methionine- or N-acetylcysteine-induced developmental toxicity is confined to early stages. A role for oxidative activity is suggested in modulating cell differentiation and embryogenesis, consistent with antioxidant-induced damage to early life stages.

Cloning and transcriptional analysis of the lipA (lipoic acid synthetase) gene from Rhizobium etli.

We report here the isolation of a Rhizobium etli gene involved in lipoic acid metabolism, the lipA gene, which complements a lipA mutant strain of Escherichia coli. A promoter region (lipAp) was mapped immediately upstream of lipA and two in vivo transcription initiation sites were identified, preceded by sequences showing some homology to the -10/-35 promoter consensus sequences. The activity of the lipAp was found not to be regulated either by the carbon source or by the addition of lipoic acid. Moreover, quantitative analysis of the lipA transcript by RNase protection assays indicated its down-regulation during entry into stationary phase.