Glioblastoma cells inhibit astrocytic p53-expression favoring cancer malignancy.

The tumor microenvironment has a dynamic and usually cancer-promoting function during all tumorigenic steps. Glioblastoma (GBM) is a fatal tumor of the central nervous system, in which a substantial number of non-tumoral infiltrated cells can be found. Astrocytes neighboring these tumor cells have a particular reactive phenotype and can enhance GBM malignancy by inducing aberrant cell proliferation and invasion. The tumor suppressor p53 has a potential non-cell autonomous function by modulating the expression of secreted proteins that influence neighbor cells. In this work, we investigated the role of p53 on the crosstalk between GBM cells and astrocytes. We show that extracellular matrix (ECM) from p53(+/-) astrocytes is richer in laminin and fibronectin, compared with ECM from p53(+/+) astrocytes. In addition, ECM from p53(+/-) astrocytes increases the survival and the expression of mesenchymal markers in GBM cells, which suggests haploinsufficient phenotype of the p53(+/-) microenvironment. Importantly, conditioned medium from GBM cells blocks the expression of p53 in p53(+/+) astrocytes, even when DNA was damaged. These results suggest that GBM cells create a dysfunctional microenvironment based on the impairment of p53 expression that in turns exacerbates tumor endurance.

Effect of increasing zinc sulphate concentration during in vitro maturation of bovine oocytes.

The objective was to investigate the effects of supplementary zinc (Zn) during in vitro maturation (IVM) of bovine oocytes. The DNA damage in cumulus cells was low with supplemental Zn concentrations of 1.1 and 1.5 µg/mL in the IVM medium (mean ± SEM index of DNA damage was 67.52 ± 9.32, 68.52 ± 13.34, 33.80 ± 4.89, and 34.65 ± 7.92 for supplementation with 0, 0.7, 1.1, and 1.5 µg/mL Zn, respectively; P < 0.01). Total glutathione concentrations did not differ following Zn supplementation of 1.1 and 1.5 µg/mL (3.7 ± 0.4 vs. 4.0 ± 0.5 pmol, respectively, in oocytes; and in cumulus cells, 0.5 ± 0.04 nmol/10(6) cells, combined for both treatments), but were greater (P < 0.01) than supplementation with 0.7 µg/mL (1.8 ± 0.5 pmol in oocytes and 0.2 ± 0.02 nmol/10(6) cumulus cells). Cleavage rate increased (P < 0.05) when Zn was added to the IVM medium at any concentration (67.16 ± 1.17, 73.15 ± 1.15, 74.05 ± 1.23, and 72.76 ± 0.74 for 0, 0.7, 1.1, and 1.5 µg/mL Zn). For these concentrations, subsequent embryo development to the blastocyst stage was 17.83 ± 2.15, 21.95 ± 0.95, 27.65 ± 1.61, and 30.33 ± 2.78%, highest (P < 0.01) in oocytes matured with 1.5 µg/mL Zn. There was an increase (P < 0.05) in mean cell number per blastocyst obtained from oocytes matured with 1.1 and 1.5 µg/mL Zn relative to 0 Zn (IVM alone) and 0.7 µg/mL Zn. In conclusion, Zn during oocytes maturation significantly affected intracellular GSH content and DNA integrity of cumulus cells, and improved preimplantational embryo development. We inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Zn concentrations.

Metabolic requirements associated with GSH synthesis during in vitro maturation of cattle oocytes.

Glutathione (GSH) concentration increases in bovine oocytes during in vitro maturation (IVM). The constitutive amino acids involved in GSH synthesis are glycine (Gly), glutamate (Glu) and cysteine (Cys). The present study was conducted to investigate the effect of the availability of glucose, Cys, Gly and Glu on GSH synthesis during IVM. The effect of the amino acid serine (Ser) on intracellular reduced/oxidized glutathione (GSH/GSSG) content in both oocytes and cumulus cells was also studied. Cumulus-oocyte complexes (COC) of cattle obtained from ovaries collected from an abattoir were matured in synthetic oviduct fluid (SOF) medium containing 8 mg/ml bovine serum albumin-fatty acid-free (BSA-FAF), 10 microg/ml LH, 1 microg/ml porcine FSH (pFSH) and 1 microg/ml 17 beta-estradiol (17beta-E2). GSH/GSSG content was measured using a double-beam spectrophotometer. The COC were cultured in SOF supplemented with 1.5mM or 5.6mM glucose (Exp. 1); with or without Cys+Glu+Gly (Exp. 2); with the omission of one constitutive GSH amino acid (Exp. 3); with 0.6mM Cys or Cys+Ser (Exp. 4). The developmental capacity of oocytes matured in IVM medium supplemented with Cys and the cell number per blastocyst were determined (Exp. 5). The results reported here indicate (1) no differences in the intracellular GSH/GSSG content at any glucose concentrations. Also, cumulus cell number per COC did not differ either before or after IVM (Exp. 1). (2) Glutathione content in oocytes matured in SOF alone were significantly different from oocytes incubated with SOF supplemented with Cys+Glu+Gly (Exp. 2). (3) Addition of Cys to maturation medium, either with or without Gly and Glu supplementation resulted in an increase of GSH/GSSG content. However, when Cys was omitted from the IVM medium intracellular GSH in oocytes or cumulus cells was less but not significantly altered compared to SOF alone (Exp. 3). (4) Glutathione content in both oocytes and cumulus cells was significantly reduced by incubation with 5mM Ser (Exp.4). (5) There was a significant increase in cleavage and blastocyst rates when Cys was added to maturation medium. In contrast, the cleavage, morula and blastocyst rates were significantly different when 5mM Ser was added to maturation media. There was also a significant difference in mean cell number per blastocyst, obtained from oocytes matured with 5mM Ser (Exp. 5). This study provides evidence that optimal embryo development in vitro is partially dependent on the presence of precursor amino acids for intracellular GSH production. Moreover, the availability of Cys might be a critical factor for GSH synthesis during IVM in cattle oocytes. Greater Ser concentration in IVM medium altered "normal" intracellular GSH in both oocytes and cumulus cells with negative consequences for subsequent developmental capacity.

Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro.

In vitro use of arresters of meiosis could improve cytoplasmic maturation of immature oocytes by controlling the period of prophase I. Phosphodiesterases (PDE) are responsible for the breakdown and concomitant inactivation of the cyclic nucleotides cAMP and cGMP and are implicated in the regulation of oocyte meiotic maturation. Selective inhibitors of phosphodiesterase type 3 (PDE3) prevent meiotic resumption of mammalian oocytes. This study evaluated the impact of meiosis arrest by PDE3 inhibitor, Org 9935, on developmental competence of geminal vesicle (GV)-stage oocytes from small antral follicles. Cumulus-oocyte complexes (COC), retrieved from antral follicles 24 h after eCG exposure and cultured in the presence of PDE3 inhibitor (10 microM) for an additional 24 h, remained arrested in the meiotic prophase. The GV configuration of oocytes before and after the arrest by PDE3 inhibitor was examined. After the period of meiosis arrest, a significantly increased proportion of oocytes had acquired a nucleolus surrounded by a condensed chromatin rim at the GV, which is a morphological correlate of transcriptional repression. Removal of inhibitor resulted in 90.6% +/- 8.3% of oocytes with the first polar body extruded. Fertilization was significantly improved in oocytes that had been arrested compared with oocytes collected 24 h after eCG and undergoing in vitro maturation immediately. Embryonic preimplantation and live offspring rates of arrested oocytes were higher, although not significantly, than those of nonarrested oocytes. These results suggest that a temporal block of meiosis by PDE3 inhibitor promotes developmental competence of mice oocytes retrieved from small antral follicles.

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine.

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.

Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations.

The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol + glycerol (Eg + Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg + Gly + 0.1 M sucrose group had a greater rate as compared with Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6 + Day 7 embryos; and, Eg + Gly + 0.3 M sucrose group had a greater rate as compared with the Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in evaluations of Day 6 and Day 6 + Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg + Gly + 0.1 M and Eg + Gly + 0 M sucrose groups and the Eg + Gly + 0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg + Gly + 0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs.

Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition.

Supraphysiological oxygen tension during embryo culture can generate reactive oxygen species (ROS), which can induce apoptosis. Antioxidants such as thiol compounds (cysteine, cysteamine) can be used to prevent ROS damage to the embryo. The purpose of this study was to evaluate the prevalence of apoptosis during bovine embryo development and to evaluate the effect of the presence or absence of cysteine 0.6 mM in modified synthetic oviduct fluid (mSOF) on in vitro produced cattle embryos cultured under two different oxygen tensions (5% O2 versus 20% O2). Effects were assessed by checking embryo development at Days 7, 8 and 9 and by evaluating Day 9 hatched blastocysts for differentiation by means of differential staining and for apoptosis by means of TUNEL-assay. Apoptotic cells were present in 94% of Day 7 blastocysts and in 100% of Days 8 and 9 blastocysts. Cysteine addition affected Day 8 blastocyst rates in a negative way (P < 0.05) regardless of the oxygen tension. In fact, cysteine addition to the mSOF culture medium had a negative effect upon embryo development in terms of blastocyst rates, hatching rates and apoptotic cell ratio. Embryos cultured under 5% O2 in the presence of cysteine, however, possessed significantly higher numbers of ICM cells. This finding corroborates the theoretical assumption that antioxidants are beneficial for ICM development.

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production.

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.

Efecto de la estimulación de la síntesis de glutation durante la maduración de oocitos de bovinos y cultivo de los embriones in vitro en la tasa de desarrollo y calidad de los embriones / Effect of stimulation of glutathione synthesis during in vitro maturation of bovine oocytes and in vitro embryo culture on the development rate and embryo quality

Objetivo: El desarrollo de un sistema de maduración (MIV) y cultivo (CIV) in vitro de oocitos humanos es importante. El uso de oocitos humanos en investigación es problemático. Los oocitos de bovinos han sido propuestos como el modelo más conveniente. Se llevaron a cabo experimentos donde se evaluó el efecto que tiene la estimulación de la síntesis de glutation (GSH) durante la MIV y el CIV sobre el desarrollo embrionario y la calidad de los mismos. Materiales y Métodos: Los oocitos provenientes de ovarios de matadero fueron madurados, fertilizados, cultivados y congelados in vitro. La síntesis de glutation fue estimulada con cisteamina. La tasa de desarrollo y calidad de los embriones se estudió en 5 grupos: MIV y CIV (Día 2, embriones de 2 a 6 células) sin suplementación de cisteamina (Cist) (Grupo Control) (A); MIV suplementado con 100 mM de Cist (MIV-100) y el CIV sin suplementación (B); MIV-100 y CIV suplementado con 25 µM de Cist (C); o 50 µM de Cist (D); o con 100 µM de Cist (E). Se realizaron 7 réplicas con 1.374 oocitos. Los datos transformados se analizaron mediante ANOVA y test de Tukey. Resultados: El desarrollo de los embriones en los grupos B, C y D fueron significativamente superiores al grupo control (A). El grupo D fue el mejor (P<0.05). Además, el grupo D presentó los mejores resultados de sobrevida y eclosión embrionaria luego del congelamiento, comparado con el grupo A. Discusión: Los resultados demuestran que la estimulación de la síntesis de GSH durante la MIV y el CIV de oocitos bovinos mejora las tasas de desarrollo de los embriones y su calidad. Estos resultados nos muestran el papel preponderante que cumple el metabolismo del GSH durante la maduración citoplasmática y el desarrollo de los embriones

Efecto de la estimulación de la síntesis de glutation durante la maduración de oocitos de bovinos y cultivo de los embriones in vitro en la tasa de desarrollo y calidad de los embriones / Effect of stimulation of glutathione synthesis during in vitro maturation of bovine oocytes and in vitro embryo culture on the development rate and embryo quality

Objetivo: El desarrollo de un sistema de maduración (MIV) y cultivo (CIV) in vitro de oocitos humanos es importante. El uso de oocitos humanos en investigación es problemático. Los oocitos de bovinos han sido propuestos como el modelo más conveniente. Se llevaron a cabo experimentos donde se evaluó el efecto que tiene la estimulación de la síntesis de glutation (GSH) durante la MIV y el CIV sobre el desarrollo embrionario y la calidad de los mismos. Materiales y Métodos: Los oocitos provenientes de ovarios de matadero fueron madurados, fertilizados, cultivados y congelados in vitro. La síntesis de glutation fue estimulada con cisteamina. La tasa de desarrollo y calidad de los embriones se estudió en 5 grupos: MIV y CIV (Día 2, embriones de 2 a 6 células) sin suplementación de cisteamina (Cist) (Grupo Control) (A); MIV suplementado con 100 mM de Cist (MIV-100) y el CIV sin suplementación (B); MIV-100 y CIV suplementado con 25 AM de Cist (C); o 50 AM de Cist (D); o con 100 AM de Cist (E). Se realizaron 7 réplicas con 1.374 oocitos. Los datos transformados se analizaron mediante ANOVA y test de Tukey. Resultados: El desarrollo de los embriones en los grupos B, C y D fueron significativamente superiores al grupo control (A). El grupo D fue el mejor (P<0.05). Además, el grupo D presentó los mejores resultados de sobrevida y eclosión embrionaria luego del congelamiento, comparado con el grupo A. Discusión: Los resultados demuestran que la estimulación de la síntesis de GSH durante la MIV y el CIV de oocitos bovinos mejora las tasas de desarrollo de los embriones y su calidad. Estos resultados nos muestran el papel preponderante que cumple el metabolismo del GSH durante la maduración citoplasmática y el desarrollo de los embriones (AU)

Trypsin inhibitor from Dimorphandra mollis seeds: purification and properties.

A trypsin inhibitor from Dimorphandra mollis seeds was isolated to apparent homogeneity by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange and affinity chromatographic techniques. SDS-PAGE analysis gave an apparent molecular weight of 20 kDa, and isoelectric focusing analysis demonstrated the presence of three isoforms. The partial N-terminal amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz family of inhibitors. This inhibitor, which inhibited trypsin activity with a Ki of 5.3 x 10(-10) M, is formed by a single polypeptide chain with an arginine residue in the reactive site.

The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine.

Supplementation of IVM medium with cysteamine, beta-mercaptoethanol, cysteine and cystine induced bovine oocyte glutathione (GSH) synthesis, but only the effect of cysteamine on the developmental competence of these oocytes was tested. During IVM of sheep oocytes, cysteamine but not beta-mercaptoethanol increased embryo development. However, it is not known how long the high intracellular oocyte GSH levels obtained after IVM with thiol compounds, can be maintained. Thus, the present study was carried out to evaluate the effects of supplementing maturation medium with 100 microM beta-mercaptoethanol, 0.6 mM cysteine and 0.6 mM cystine on 1) intracellular GSH level after IVM, 2) after IVF, 3) in 6 to 8-cell embryos and 4) on embryo development. In oocytes after IVM and in presumptive zygotes after IVF, intracellular GSH levels were significantly higher in the treated groups (P < 0.05). While, GSH content in 6 to 8-cell embryos was similar among treatment groups (P > 0.05). Differences in cleavage rates and the percentage of embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) for treated oocytes than for those matured in the control medium. We conclude from the results that the high intracellular GSH levels after induction of GSH synthesis in bovine IVM by thiol compounds remain during IVF and are still present at the beginning of IVC, improving developmental rates. Moreover, the results indicate that this metabolic pathway is an important component of the cytoplasmic maturation process that affects the subsequent steps of in vitro embryo production.

Cumulus expansion during in vitro maturation of bovine oocytes: relationship with intracellular glutathione level and its role on subsequent embryo development.

Glutamine (GLN) is a metabolic precursor for hexosamine synthesis and its inclusion in culture medium has been reported to improve cumulus expansion. Glutamine and cysteine share the same transport system. Excess external GLN may act as a competitive inhibitor for the uptake of cysteine and stimulate loss of cellular cysteine, interfering this with GSH synthesis. Experiments were designed to evaluate the effect of 1-3 mM GLN during in vitro maturation (IVM) on bovine-cumulus expansion, intracellular GSH levels in both oocytes and cumulus cells, and subsequent embryo development up to blastocyst stage. Also, GSH content was measured in 6- to 8-cell embryos and a possible relationship between cumulus expansion and GSH synthesis was studied. Intact cumulus cell-oocyte complexes were incubated for 24 hr and cumulus expansion was measured by a computerized image-digitizing system either before or after IVM. IVM/IVF bovine oocytes were cultured up to 6- to 8-cell stage embryos for assessment of GSH content or for 8 days up to blastocyst stage for embryo development. The measurement of total GSH content was performed by an enzymatic method in oocytes, cumulus cells and 6- to 8-cell embryos. The maximal expansion was achieved by addition of 2 mM GLN without affecting GSH levels, in both oocytes and cumulus cells. At 3 mM, the degree of cumulus expansion was lower and the GSH levels decreased. The addition of 2 mM GLN improves cleavage and blastocyst rates, whereas no differences were found between O, 1, and 3 mM GLN. Moreover, the GSH content in 6- to 8-cell embryos was similar at any GLN concentrations. In order to study the relationship between GSH and cumulus expansion: 6-diazo-5-oxo-1-norleucine (DON), an inhibitor of hexosamine synthesis, or buthionine sulfoximide (BSO), an inhibitor of GSH synthesis, either alone or with GLN was added to IVM medium. GSH level was not affected by the presence of DON. However, the degree of cumulus expansion was reduced in the presence of BSO. In conclusion, bovine oocytes matured in the presence of 2 mM GLN improve their capacity for subsequent embryo development. Nevertheless, GSH level was altered when GLN was added to IVM medium at a high concentration with a reduction in the degree of cumulus expansion. This study provides evidence that optimal cumulus expansion in vitro is partially dependent on hexosamine production and intracellular GSH content.

Effect of hyaluronic acid on development of in vitro produced bovine embryos.

The present study was carried out to evaluate the effect of hyaluronic acid (HA) added to the culture medium on bovine embryo development to the blastocyst stage as well as embryo quality and viability after freezing and thawing. In vitro matured and fertilized (IVM/IVF) bovine oocytes from slaughterhouse ovaries were cultured for 8 d in SOFm supplemented with 4 mg/mL fatty acid-free BSA, either in the absence or presence of 1 or 0.5 mg/mL HA. There was a significant increase in blastocyst yield in the presence of 1 mg/mL HA (P < 0.01), whereas 0.5 mg/mL HA was ineffective. Cleavage rate and mean number of days to blastocyst formation were unaffected by HA at any concentration. At 1 mg/mL, HA did not affect either post-freeze survival of Grade 1 and 2 blastocysts or the number of nuclei per blastocyst. Supplementation with HA at 1 mg/mL also significantly enhanced embryo development up to the blastocyst stage (P < 0.05) in a chemically-defined culture medium without a protein source. It is concluded that supplementation of both semi-defined and defined culture media with 1 mg/mL HA improves the development of IVM/IVF bovine embryos to the blastocyst stage, without affecting embryo quality and post-freeze survival. These results open the possibility of including HA in culture media in order to increase the efficiency of in vitro blastocyst production from in vitro-matured bovine oocytes.

In vitro evaluation and pregnancy rates after vitrification of in vitro produced bovine embryos.

The efficacy of different vitrification solutions to cryopreserve in vitro-produced bovine blastocysts was evaluated based on in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, 2 vitrification solutions were compared: propylene glycol + glycerol (Pg + Gly) and ethylene glycol + Ficoll + sucrose (EFS). Differences in the overall development and hatching rates in favor of EFS were found (56.4 vs 33.3% and 35.4 vs 13.3%; P < 0.05). In the second experiment, 3 vitrification solutions were compared: EFS, modified EFS (EFSm) and ethylene glycol + glycerol (Eg + Gly). The vitrification solutions EFSm and Eg + Gly yield higher hatching rates than did EFS (57.7 vs 59.6 vs 35.7%; P < 0.05). The last experiment was designed to compare in vivo 2 vitrification solutions: EFSm and Eg + Gly. There were no differences between them based on the results obtained after transfer (35.2 vs 43.7%). The vitrification solutions EFSm and Eg + Gly have resulted in good pregnancy rates. These results demonstrated that vitrification can be used successfully in the cryopreservation of in-vitro produced bovine embryos, and it might be considered for use in commercial programs.

Prevalence of serotypes and antimicrobial resistance of streptococcus pneumoniae strains isolated from Brazilian children with invasive infections. Pneumococcal Study Group in Brazil for the SIREVA Project. Regional System for Vaccines in Latin America.

A laboratory surveillance study was developed in Brazil in 1993 to determine capsular types and antimicrobial susceptibility of Streptococcus pneumoniae strains. By studying 360 strains isolated from children with invasive infections in three different cities, 8 out of 34 types were identified as being the most prevalent and considered as the reference group for further analyses. This group comprised 77.7% of all strains studied, and includes the types 1, 5, 6A/B, 9V, 14, 19F, 19A, and 23F. The prevalence of this reference group was significantly higher among strains isolated from children with pneumonia than meningitis. Similarly, this group was more prevalent among strains isolated from children 3 to 6 years of age than from children under 2 years of age. Most strains (78.6%) were found to be susceptible to penicillin and only 1.4% showed high resistance to this antibiotic. However, intermediate resistance to penicillin was detected in 20% of the strains. This laboratory surveillance will be maintained and extended to other cities of Brazil to better define and monitor the trends of pneumococcal infections for proper control and prevention.

Glutathione synthesis during in vitro maturation of bovine oocytes: role of cumulus cells.

Glutathione (GSH) synthesis during in vitro maturation (IVM) has been shown to play an important role in embryo development. The present study was carried out to evaluate the role of cumulus cells in GSH synthesis during IVM of bovine oocytes in the presence of cystine, cysteine, the cysteine analogue N-acetylcysteine, and cysteamine. For this purpose, cumulus-oocyte complexes (COCs), denuded oocytes (DOs), and DOs in coculture with a cumulus cell monolayer were used. An increase in GSH level stimulated by cystine was observed only in the presence of cumulus cells, either with COCs or in DOs matured on a coculture monolayer. Addition of cysteine and cysteamine to IVM medium increased GSH levels in COCs and DOs. N-Acetylcysteine increased GSH levels only in DOs. Moreover, cumulus cells contributed to the stimulatory effect exerted by cysteine and cysteamine on GSH synthesis in COCs. These results indicate that cumulus cells during IVM play an important role in oocyte GSH synthesis, allowing the oocytes to use cystine and contributing to the stimulatory effect exerted by cysteine and cysteamine. In addition, these results demonstrate that IVM medium supplemented with cysteine or cysteamine increased GSH content in oocytes without cumulus mass (DO) and in the absence of a cumulus cell monolayer. This may be useful to increase the efficacy of IVM of those oocytes having few cumulus cell layers, in a system without coculture.

Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability.

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of beta-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, beta-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing.

In vitro production of sheep embryos using laparoscopic folliculocentesis: alternative gonadotrophin treatments for stimulation of oocyte donors.

Three different gonadotrophin regimens for the stimulation of donors for laparoscopic folliculocentesis were tested in a total of 142 ewes. The recovered oocytes were subjected to in vitro maturation, fertilization, and culture (IVM/IVF/IVC) for 7 d using standard procedures for sheep. The estrous cycles of all ewes were synchronized using intravaginal sponges containing 60 mg of medroxyprogesterone acetate (MPA) inserted for 14 d. In Experiment 1, all ewes were superovulated with a total dose of 125 IU FSH and 125 IU LH. One-half of the ewes received the gonadotrophin treatment in 4 decreasing doses at 12-h intervals starting 48 h before follicle aspiration (Control), while the other half received the total dose in a single injection at -24 h before collection (Oneshot). There were no significant differences between treatments for recovery rate (81.6 +/- 5.3 vs 77.4 +/- 10.3), cleavage rate (60.6 +/- 20.8 vs 61.4 +/- 23.4), or normal development to the blastocyst stage (20.8 +/- 18.2 vs 13.1 +/- 10.3). However, a higher percentage of ewes produced at least 1 normal blastocyst in the Control group (56.4 vs 31.6%; P < 0.05). In Experiment 2, the control regimen was repeated in half of the ewes, while the remainder were treated with half of the FSH total dose plus 500 IU eCG in a single injection at -24 h before oocyte collection (Oneshot-eCG). The recovery rate (80.9 +/- 5.6 vs 73.3 +/- 15.3), cleavage rate (76.8 +/-19.9 vs 79.7 +/- 22.6), normal development to blastocysts (19.2 +/- 15.3 vs 23.3 +/- 10.7), and percentage of ewes producing at least 1 normal blastocyst (55.9 vs 51.6%) did not differ between treatments. The large variability observed between ewes in the production of normal blastocysts is comparable to that observed with standard MOET procedures, in which a proportion of donors fail to produce good embryos. With the in vitro procedures described here, we were able to produce normal embryos from more than half of the treated ewes, indicating that the technology is useful for the multiplication of genetically valuable animals affected by temporary or irreversible infertility.

Effect of cysteamine on glutathione level and developmental capacity of bovine oocyte matured in vitro.

The present study was carried out to evaluate if the addition of cysteamine to the culture medium during in vitro maturation of bovine oocytes increased the glutathione (GSH) levels in the mature oocytes, and if these changes may promote an improvement on in vitro development to the blastocyst stage. Follicular oocytes from slaughterhouse ovaries were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and O (control), 25, 50, or 100 muM of cysteamine for 24 hr. After in vitro maturation the oocytes were fertilized and cultured for 8 days. The percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.01) for oocytes matured in medium containing 100 muM of cysteamine than for those matured in control medium. Moreover, the intracellular GSH levels were increased (P < 0.05) in oocytes matured with 100 muM of cysteamine with respect to control. No differences were observed in maturation and cleavage rates, and in the mean cell numbers per blastocyst among treatments (P > 0.05). These results indicate that the addition of thiol compounds such as cysteamine to maturation medium increases the efficiency of in vitro blastocyst production from immature bovine oocytes. The higher levels of GSH in oocytes matured in the presence of cysteamine suggest that the beneficial effects of cysteamine on in vitro maturation and subsequent development after in vitro fertilization are mediated by GSH.