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 0, 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. Mol. Reprod. Dev. 51:76–83, 1998. © 1998 Wiley-Liss, Inc.     

The role of the visceral yolk sac in hyperglycemia-induced embryopathies in mouse embryos in vitro.

The adverse developmental effects of hyperglycemia to rodent embryos have been shown using whole embryo culture. Although, a mechanism by which hyperglycemia-induced effects occur is unknown, recent work has focused on the visceral yolk sac as a potential target tissue. Therefore, we have evaluated the developmental effects of hyperglycemia in early head fold stage mouse embryos in vitro and assessed the histiotrophic function of the visceral yolk sac. As has been previously shown in rodents, hyperglycemia produced neural tube closure defects in a concentration dependent manner at 33, 50, and 67 mM glucose using a 44 h exposure period. However, exposure times between 6 and 12 h were sufficient to alter embryonic development when the glucose concentration was 50 or 67 mM. In contrast, early somite stage embryos (4-6 somite stage) appear to be less sensitive to dysmorphogenesis and a 48 h exposure to 67 mM glucose but not 33 or 50 mM also produced neural tube defects. Hyperglycemia (67 mM) did not alter the uptake of 35S-methionine and 35S-cysteine-labeled hemoglobin (35S-Hb) in the visceral yolk sac (VYS) in early headfold staged embryos. However, the accumulation of 35S in the embryo was reduced by 16-18% at glucose concentrations of 50 or 67 mM during the last 12 h of a 44 h exposure period. No effect on VYS uptake or embryonic accumulation of 35S-labeled products was observed at shorter exposure periods (12-24 and 24-36 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of glutathione level in human melanoma cells: effect of N-acetyl-L-cysteine and its analogues

The effects of N-acetyl-L-cysteine (L-NAC), N,N-diacetyl-L-cystine (oxidized form of L-NAC) and N-acetyl-D-cysteine on the intracellular glutathione (GSH) level and their toxicity were investigated in the human melanoma cell culture IGR1. L-NAC applied in 3 mM concentration for 24 hr decreased; when applied for 48 hr it did not alter the intracellular GSH level. Treatment with 1 mM L-NAC for 24 hr had no effect on cellular glutathione, whereas the same concentration applied for 48 hr resulted in an increase in the level of GSH. Both concentrations also induced cell injury as determined by protein assay and trypan blue staining. N,N-diacetyl-L-cystine (0.5 and 1.5 mM, 24 hr) induced a decrease in cellular glutathione content without any apparent cell toxicity. D-NAC (1 and 3 mM, 24 hr) did not influence the GSH level of the melanoma cells; however, it had toxic effects resulting in cell loss.     

Cadherin mRNAs during rat embryo development in vivo and in vitro.

Whole rat embryo cultures are being used in increasing numbers of laboratories to study the mechanisms by which teratogens disturb development. The development of early somite stage embryos in vitro is very similar morphologically to that in vivo, yet few biochemical comparisons have been made. The purpose of this study was to determine the steady-state mRNA concentrations of a family of Ca(2+)-dependent cell adhesion molecules, the cadherins, during rat embryonic development in vivo and in vitro. Embryos and yolk sacs were collected on days 10, 11, and 12 of gestation (in vivo); they were also obtained from day 10 embryos after growth in culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). Total RNAs isolated from embryos and yolk sacs were studied by Northern blot analysis using specific cDNA probes for three cadherins, E-cadherin, N-cadherin, and P-cadherin. Although E-cadherin mRNA was detected in embryos, it was present at much higher concentrations in yolk sacs. In addition, multiple species of E-cadherin mRNA ranging from 3.0 to 13 kb were detected. Interestingly, the concentration of the major 4.5-kb E-cadherin mRNA species in yolk sac after 45 hr in culture was increased 2.8-fold over that on day 12 of gestation in vivo. Second, two species (4.3 and 3.5 kb) of N-cadherin mRNA were detected, almost exclusively in embryos. In yolk sac, N-cadherin mRNA was detected only after 45 hr in culture. Third, P-cadherin mRNA was detected as a single 3.5-kb species, mainly in embryos. P-cadherin mRNA concentrations in yolk sac after 45 hr in culture were 5.6-fold higher than in vivo. Thus, these results demonstrate that there is a differential distribution of cadherin mRNAs in rat embryos and yolk sacs. Further, there appear to be multiple species of mRNAs for E-cadherin and N-cadherin. Finally, while whole embryo culture in vitro did not significantly alter the steady-state concentrations of cadherin mRNAs in the embryo, these concentrations were dramatically increased in the yolk sac.     

Relationships between formaldehyde metabolism and toxicity and glutathione concentrations in isolated rat hepatocytes

The metabolism and toxicity of formaldehyde (CH2O) in isolated rat hepatocytes was found to be dependent upon the intracellular concentration of glutathione (GSH). Using hepatocytes depleted of GSH by treatment with diethyl maleate (DEM), the rate of CH2O (5.0 mM) disappearance was significantly decreased. Formaldehyde decreased the concentration of GSH in hepatocytes, probably by the extrusion of the CH2O-GSH adduct, S-hydroxymethylglutathione. Formaldehyde toxicity was potentiated in cells pretreated with 1.0 mM DEM as measured by the loss of membrane integrity (NADH stimulation of lactate dehydrogenase (LDH) activity) and an increase in lipid peroxidation (formation of thiobarbituric acid-reactive compounds). This potentiation of toxicity was both CH2O concentration-dependent and time-dependent. There was an excellent correlation between the increase in lipid peroxidation and the decrease in cell viability. L-Methionine (1.0 mM) both protected the cells from toxicity caused by the combination of 8.0 mM CH2O and 1.0 mM DEM and increased the cellular GSH concentration. The antioxidants, ascorbate, butylated hydroxytoluene (BHT) and alpha-tocopherol (10, 25 and 125 microM), all exhibited dose-dependent protection against toxicity produced by 8.0 mM CH2O and 1.0 mM DEM. At toxic concentrations of CH2O (10.0-13.0 mM), administered by itself, lipid peroxidation did not increase concomitantly with the decrease in cell viability and the addition of antioxidants (125 microM) did not influence CH2O toxicity. These results suggest that CH2O toxicity in GSH-depleted hepatocytes may be mediated by free radicals as a result of the effect of CH2O on a critical cellular pool of GSH. However, cells with normal concentrations of GSH are damaged by CH2O by a different mechanism.     

Buthionine sulfoximine embryotoxicity is associated with prolonged AP-1 activation

BACKGROUND: Many teratogens induce oxidative stress, altering redox status and redox signaling; this has led to the suggestion that developmental toxicants act by disturbing redox status. The goal of these studies was to determine the consequences of altering glutathione homeostasis during organogenesis on embryo development, total DNA methylation, and activator protein-1 (AP-1) DNA binding activity and gene expression. METHODS: Gestational day 10.5 rat embryos were cultured in vitro for up to 44 hour in the presence of L-buthionine-S,R-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamyl-cysteine synthetase, the rate limiting step in glutathione biosynthesis. Effects of BSO on total, oxidized and reduced glutathione, embryo development, DNA methylation, AP-1 DNA binding activity and gene expression were investigated. RESULTS: Significant depletion of glutathione by BSO was first noted at 6 hr in the embryo and at 3 hr in the yolk sac; total glutathione in the conceptus was depleted to the same extent after treatment with either 0.1 or 1.0 mM BSO. Exposure to 0.1 mM BSO did not cause a significant increase in embryotoxicity, although some impairment of growth and development was observed. In contrast, exposure to 1.0 mM BSO severely inhibited growth and development, significantly increasing the incidence of swollen hindbrains and of blebs in the forebrain, limb and maxillary regions. No significant treatment-related differences in total DNA methylation were observed. Interestingly, AP-1 DNA binding activity was similar in control and 0.1 mM BSO-treated conceptuses; however, exposure to 1.0 mM BSO increased AP-1 DNA binding at 6, 24, and 44 hr. The expression of several AP-1 family genes and of gamma-glutamylcysteine synthetase was induced in embryos cultured with 1.0 mM BSO. CONCLUSION: Exposure of embryos in vitro to BSO at a concentration that was embryotoxic induced prolonged AP-1 DNA binding activity and altered gene expression. These data suggest that AP-1 induction may serve as a biomarker of embryo stress.     

Hepatic glutathione concentrations linked to ethionine toxicity in rats

1. The effect of injected ethionine on liver GSH concentrations was studied in male and female rats. 2. Liver GSH concentrations were markedly and consistently decreased in 5hr. and elevated within 24-44hr. By 72hr. the amount of liver GSH of ethionine-poisoned rats had the same values as saline-injected control rats. At no time was erythrocyte GSH concentration affected by ethionine. 3. The concentrations of non-protein thiol compounds, glycogen and ATP were also significantly decreased when the rats were killed 5hr. after ethionine injection. 4. ATP, adenine or methionine did not prevent the decrease of liver GSH produced by ethionine. From these and other observations we conclude that ethionine is not an antagonist of methionine with respect to GSH metabolism. 5. The metabolic relationship between ethionine toxicity and liver GSH concentration is briefly discussed.     

Exogenous gamma-glutamyl cycle compounds supplemented to in vitro maturation medium influence in vitro fertilization, culture, and viability parameters of porcine oocytes and embryos

High concentrations of intracellular glutathione (GSH) enhance in vitro production of porcine embryos. Objectives were: (1) to determine the effects of gamma-glutamyl cycle compound supplements to the IVM medium on IVF and IVC; and (2) to evaluate embryo viability. Porcine oocytes were matured in NCSU 23 medium supplemented with either l-cysteine (3.3 mM), l-cysteamine (150 P < 0.05microM), l-cysteine and l-cystemaine, l-glycine (1, 2.5, or 5 mM), l-glutamate (1, 2.5, or 5 mM), l-alpha-aminobutyrate (3.3mM), beta-mercaptoethanol (BME) (25 microM), l-cysteine and BME, or l-alpha-aminobutyrate and BME. Increases (P < 0.05) in GSH concentrations were observed using l-cysteine, 1.0 mM l-glutamate, l-alpha-aminobutyrate, and l-alpha-aminobutyrate with BME. Oocytes matured with l-alpha-aminobutyrate and BME had a lower (P < 0.05) occurrence of polyspermy during IVF compared to controls and a greater percentage (P < 0.05) of embryos reaching the blastocyst stage compared to other treatment groups. For Objective 2, oocytes were matured in NCSU 23 or NCSU 23 supplemented with l-alpha-aminobutyrate with BME. Embryo cell death was determined using an Annexin V-FITC assay. Supplementation had no effect on the time of cell death. Embryo mortality was increased (P < 0.05) from 24 to 42 h post-IVF, with the greatest occurrence around 36 h. In conclusion, supplementing l-alpha-aminobutyrate and BME into the IVM medium increased intracellular GSH concentrations, decreased the occurrence of polyspermy during IVF, and increased embryo development parameters during IVC, but did not affect cell death during embryo development. The onset of cell death occurred from 24 to 42 h post-IVF, with the greatest occurrence around 36 h post-IVF.     

Effects on in vitro embryo development and intracellular glutathione content of the presence of thiol compounds during maturation of prepubertal goat oocytes

The low number of embryos produced from in vitro matured, fertilized, and cultured (IVM-IVF-IVC) oocytes of prepubertal goat is mainly due to a low incidence of sperm head decondensation at fertilization (Martino et al., 1995: Theriogenology 43:473-485; Mogas et al., 1997: Theriogenology 48:815-829). Thiol compounds stimulate glutathione (GSH) synthesis and improve the rates of male pronucleus (MPN) formation and embryo development. The present study was carried out to determine whether supplementation of the IVM medium with 100 microM of cysteamine, 100 microM of beta-mercaptoethanol, 0.57 mM of cysteine, and 0.57 mM cystine might improve the embryo development and intracellular GSH level of prepubertal goat oocytes. After 27 hr post IVM, a sample of oocytes was frozen and the intracytoplasmic GSH content was evaluated by spectrophotometry. IVM-oocytes were inseminated with fresh semen and cultured in SOF medium. Only the addition of cysteamine to IVM media significantly improved the percentage of the morula plus blastocyst yield compared to the control group (oocytes matured in absence of thiol compounds) (22.2 vs. 6.4%, respectively; P < 0.05). The percentage of expanded blastocysts in cysteamine and control groups was 13.0 and 2.6%, respectively, and the mean cell number per blastocyst was 86.8 and 60.5, respectively. None of the other thiol compounds studied significantly improved the percentage of embryos obtained. It has been demonstrated that prepubertal goat oocytes synthesize GSH during IVM and that thiol compounds increase this GSH synthesis. In conclusion, only the addition of 100 microM of cysteamine to the maturation medium improves embryo development from prepubertal goat oocytes although all the thiol compounds used in this study increased intracellular GSH content.     

Effect of adding reduced glutathione during insemination on the development of porcine embryos in vitro

This study evaluated the effect of adding reduced glutathione (GSH) during sperm washing and insemination on the subsequent fertilization dynamics and development of IVM porcine oocytes. Follicular oocytes were matured in vitro in NCSU 23 medium with porcine follicular fluid, cysteine and hormone supplements for 22 h. They were then matured in the same medium but without hormones for another 22 h. Matured oocytes were stripped of cumulus cells and co-incubated with frozen-thawed spermatozoa for 5 h. Putative embryos were cultured in NCSU 23 with BSA for either 7 h to examine fertilization parameters or 6 d to evaluate cleavage (2 d) and blastocyst rates. In Experiment 1, GSH was added to the insemination medium at 0, 0.125, 0.25 or 0.5 mM. The presence of GSH during insemination did not affect (P>0.05) rates of penetration, polyspermy, male pronuclear formation or cleavage, but did increase (P<0.05) blastocyst formation rates when added at concentrations of 0.125 (36%) and 0.25 mM (34%) compared with that of the control (0 mM; 19%). However, the numbers of inner cell mass and trophectoderm cells of blastocysts were unaffected by GSH treatment (P>0.05). The presence of GSH during insemination was found not to significantly increase intracellular glutathione concentrations of oocytes (P>0.05). In Experiment 2, addition of GSH (0.25 mM) during sperm washing did not affect cleavage or blastocyst formation rates or cell numbers (P>0.05). In conclusion, the presence of GSH during insemination improves the developmental competence of IVM pig oocytes in a dose-dependent manner.     

Glutathione depletion modulates methanol, formaldehyde and formate toxicity in cultured rat conceptuses

The proposed use of methanol (H3COH) as an alternative to fossil fuels has prompted concern about potential health risks resulting from widespread environmental exposure. Methanol is teratogenic in rodents and, although the exact toxic species is not known, teratogenesis may result from the enzymatic biotransformation of H3COH to formaldehyde (CH2O) and formic acid causing increased biological reactivity and toxicity. A protective role for the antioxidant glutathione (GSH) has been described for H3COH, CH2O and formic acid toxicity in various biological systems but has yet to be evaluated in the developing conceptus. Whole embryo culture studies were conducted using GD 10-11 rat conceptuses to elucidate the relationship between H3COH and its metabolites and GSH status. Methanol exposure produced a decrease in normal growth parameters and a dose-dependent loss of viability. CH2O had deleterious effects on embryo growth and viability. Sodium formate (HCOONa) exposure resulted in a high mortality rate but viable embryos did not manifest any abnormalities. Methanol, CH2O, and HCOONa all produced a significant depletion of GSH in both embryo and VYS. Inhibition of GSH synthesis by L-buthionine-S,R-sulfoximine (BSO) treatment exacerbated H3COH, CH2O and HCOONa embryotoxicity. Interestingly, only H3COH/BSO and CH2O/BSO co-treatments caused increased malformation, while embryos treated with HCOONa/BSO did not produce any developmental deformities. These results implicate CH2O as the most embryotoxic H3COH metabolite, on a molar basis, in terms of causing dysmorphogenesis, alterations of normal growth parameters and embryolethality. HCOONa was selectively embryolethal and did not produce dysmorphogenesis. CH2O toxicity is potentiated by GSH depletion, indicating that GSH may be more directly involved in its detoxication in the embryo.     

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O₂ / 5% CO₂ in a rolling bottle culture apparatus at 37 °​C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.     

Influences of retrieval stages and glutathione addition on post-thaw viability of quick frozen mouse morula during in vitro culture

Effects of the embryo retrieval stages and addition of glutathione (GSH) on post-thaw development of mouse morula were evaluated in 2 consecutive experiments. In the first experiment, 1-, 2-, 3- to 4- and 5- to 8-cell stage embryos were collected and cultured to the morula stage in Whitten's medium containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). The development rate of 1-cell embryos to the morula stage was lower than that of the other stages (P<0.01). The post-thaw development rate of the morulae obtained from in vitro culture of 1-, 2-, 3- to 4-, and 5- to 8-cell embryos and from in vivo embryos (control) to the blastocyst stage was 55.5, 84.9, 87.4, 90.1 and 90.8%, respectively. The post-thaw development rate of morula obtained from in vitro produced 1-cell embryos was significantly lower than from the other stages or from the in vivo counterparts (P<0.0001). In Experiment 2, the impact of GSH supplementation of the culture medium in the presence or absence of EDTA was evaluated for embryo development to the morula stage and post-thaw survival, using in the 2 x 2 factorial design. Although EDTA supplementation increased development rates to the morulae (P<0.01) stage, GSH did not have an influence on morula development. However, the presence of either GSH or EDTA in the culture medium supported development to the blastocyst stage (P<0.01) of in vitro produced morulae. These data demonstrate that 1-cell embryos from a blocking-strain mouse cultured in vitro to the morula stage have a lower development rate following freezing and thawing than embryos collected at the 2-cell or later stages. Addition of EDTA or GSH, individually or in combination, to the culture medium may improve the development rate of morula to blastocyst stage following cryopreservation.     

Effect of the addition of glutathione and glucose to the culture medium on embryo development of IVM-IVF prepubertal goat oocytes

Our previous studies have shown that larger and more competent oocytes can be selected using the brilliant cresyl blue (BCB) test. The objective of this study was to assess, in BCB-selected oocytes, the effect on the embryo development of prepubertal goat oocytes of the addition to in vitro culture (IVC) medium of either glutathione (GSH) alone or GSH in combination with glucose. Oocytes were exposed to 26 mM BCB and were classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocyteswithout blue cytoplasm or growing oocytes (BCB-). Oocytes were matured in TCM-199 with 100 microM cysteamine. Presumptive zygotes were cultured in synthetic oviductal fluid (SOF) in the presence or absence of 1 mM glutathione (experiment 1) for 7 days (8 days post-insemination, p.i.). In experiment 2 we tested the addition to culture of 2.78 mM glucose at day 5 p.i. BCB+ oocytes showed higher percentages of nuclear maturation than the BCB- and control groups (82.6%, 55.7% and 74.7%, respectively). The percentage of polyspermic oocytes was higher in BCB- than BCB+ oocytes. Supplementation of SOF medium with 1 mM GSH did not affect embryo development but the percentage of total embryos developed after culture was higher in BCB+ oocytes than in BCB- oocytes independently of the GSH supplementation. Glucose, alone or with GSH, added at 5 days p.i. did not affect embryo development. In conclusion, prepubertal goat oocytes were unable to develop beyond the 8-cell stage embryo under the culture conditions in this study.     

Clinically Approved Iron Chelators Influence Zebrafish Mortality,Hatching Morphology and Cardiac Function

Iron chelation therapy using iron (III) specific chelators such as desferrioxamine (DFO, Desferal), deferasirox (Exjade or ICL-670), and deferiprone (Ferriprox or L1) are the current standard of care for the treatment of iron overload. Although each chelator is capable of promoting some degree of iron excretion, these chelators are also associated with a wide range of well documented toxicities. However, there is currently very limited data available on their effects in developing embryos. In this study, we took advantage of the rapid development and transparency of the zebrafish embryo, Danio rerio to assess and compare the toxicity of iron chelators. All three iron chelators described above were delivered to zebrafish embryos by direct soaking and their effects on mortality, hatching and developmental morphology were monitored for 96 hpf. To determine whether toxicity was specific to embryos, we examined the effects of chelator exposure via intra peritoneal injection on the cardiac function and gene expression in adult zebrafish. Chelators varied significantly in their effects on embryo mortality, hatching and morphology. While none of the embryos or adults exposed to DFO were negatively affected, ICL -treated embryos and adults differed significantly from controls, and L1 exerted toxic effects in embryos alone. ICL-670 significantly increased the mortality of embryos treated with doses of 0.25 mM or higher and also affected embryo morphology, causing curvature of larvae treated with concentrations above 0.5 mM. ICL-670 exposure (10 µL of 0.1 mM injection) also significantly increased the heart rate and cardiac output of adult zebrafish. While L1 exposure did not cause toxicity in adults, it did cause morphological defects in embryos at 0.5 mM. This study provides first evidence on iron chelator toxicity in early development and will help to guide our approach on better understanding the mechanism of iron chelator toxicity.     

Overcoming a permeability barrier by microinjecting cryoprotectants into zebrafish embryos (Brachydanio rerio)

The goal of this research was to examine the developmental effects on zebrafish embryos (Brachydanio rerio) when cryoprotectants were directly microinjected into the yolk. Our objectives were to: (i) determine the final concentration of propylene glycol (PG) and dimethyl sulfoxide (Me(2)SO) that the embryos could tolerate without causing teratogenic effects; (ii) determine if the toxicity of Me(2)SO could be reduced by the simultaneous presence of various proportions of amides; and (iii) examine whether this intracellular cryoprotectant incorporation could reduce the cryodamage to the yolk syncytial layer (YSL) after vitrification trials. The rationale for conducting these microinjection experiments was to overcome the permeability barrier of the YSL. Intracellular PG produced better survival than Me(2)SO (P < 0.05). Embryos tolerated both 10- and 30-nl microinjections of PG, yielding final concentrations of 2.3 and 5.0 M within the yolk, resulting in 70 +/- 3 and 35 +/- 4% survival at day 5, respectively. In similar experiments with Me(2)SO, survival was lower than PG at 60 +/- 4 and 14 +/- 4% at 2.4 and 5.2 M. Unlike other cellular systems, the presence of amides, specifically acetamide or formamide, did not reduce the toxicity of Me(2)SO in zebrafish embryos (P > 0.05). During vitrification trials, we estimated a 25% dehydration of the yolk, yielding an effective PG concentration of 5.9 M. However, the incorporation of this vitrifiable concentration of PG was not sufficient to improve the postthaw morphology of the YSL (P > 0.05). Clearly, other factors need to be examined in establishing a successful vitrification protocol for zebrafish embryos.     

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.     

Artesunate-induced depletion of embryonic erythroblasts precedes embryolethality and teratogenicity in vivo

BACKGROUND: Artesunate (ART), an artemisinin antimalarial, is embryolethal and teratogenic in rats, with the most sensitive days being 10 and 11 postcoitum (pc), respectively (Clark et al.: Birth Defects Res B 71:380-394, 2004; White et al.: Birth Defects Res A 70:265, 2004). METHODS: In this study, pregnant rats were administered a single oral dose of 17 mg/kg ART on Days 10-11 pc and conceptuses were evaluated through Day 14 pc. RESULTS: Paling of visceral yolk sacs was observed within 3-6 hr after treatment. Within 24 hr, marked paling and embryonic erythroblast depletion were observed macroscopically, which preceded malformations and embryo death, and persisted through Day 14 pc. Histologically, embryonic erythroblasts were reduced and cells showed signs of necrosis within 24 hr, were maximally depleted by 48 hr, and had partially rebounded within 3-4 days after treatment (Days 13 and 14 pc). Iron accumulation was evident in treated erythroblasts as early as 6 hr after treatment, suggesting impairment of heme synthesis. Heart abnormalities (swollen or collapsed chambers) were observed within 24 hr in approximately 25-60% of embryos and within 48 hr in 100% of embryos, correlating with histologic signs of cardiac myopathy (thinned and underdeveloped heart walls and enlarged chambers). Delays in limb and tail development occurred by Day 13 pc. Embryos were viable through Day 13 pc, but approximately 77% of embryos had died by Day 14 pc, presumably due to hypoxia and/or cardiac abnormalities. CONCLUSIONS: In summary, embryonic erythroblasts are the primary target of ART toxicity in the rat embryo after in vivo treatment, preceding embryolethality and malformations.     

Role of glutathione in reproductive tract secretions on mouse preimplantation embryo development

We investigated the hypothesis that glutathione (GSH) in reproductive tract secretions (RTS) protects the preimplantation embryo from endogenous reactive oxygen species and is important for normal development during the embryo's sensitive period when it is incapable of synthesizing GSH de novo. Mice were administered buthionine sulfoximine (BSO) to inhibit GSH synthesis and decrease GSH concentration in RTS. Embryos were then allowed to develop either in vivo or in vitro in the presence of RTS and the GSH concentration of the embryos was quantified by HPLC and embryonic development was recorded. GSH concentration in RTS did not differ over the phases of the estrous cycle, but there were significant decreases in GSH concentration on Day 2 of gestation and due to BSO treatment. Embryos allowed to develop in vivo and in vitro in RTS with decreased GSH concentration did not exhibit decreased development or GSH concentration. Oocytes exposed to BSO during maturation in vivo experienced a significant decrease in GSH concentration and an increase in percent of degenerate embryos when compared with control. These data suggest that most of the GSH in RTS does not play a critical role in normal preimplantation embryo development but that GSH stored in the oocyte during maturation has an important role in subsequent embryo development. Our studies do not exclude the possibility that GSH in RTS plays an important role in protection of the preimplantation embryo during exposure to some toxicants.     

In vitro analysis of glucose metabolism and embryonic growth in postimplantation rat embryos

The glucose metabolism and embryonic development of rat embryos during organogenesis was studied using embryo culture. Glucose uptake and embryonic growth and differentiation of 10.5-day explants (embryos + membranes) were limited by the decreasing glucose concentration, but not the increasing concentration of metabolites, in the culture media during the second 24 h of a 48 h culture. No such limitations were found on the embryonic development of 9.5-day explants during a 48 h culture although glucose uptake was slightly reduced at very low concentrations of glucose. From the head-fold stage to the 25-somite stage of development, glucose uptake was characteristic of the stage of development of the embryo and not the time it had been in culture. Embryonic growth of 9.5-day explants was similar to that previously observed in vivo. Glucose uptake by 9.5-day explants was dependent on the surface area of the yolk sac and was independent of the glucose concentration in the culture media (within the range of 9.4 to 2.5 mM). The proportion of glucose converted to lactate was 100% during the first 42h of culture then fell to about 50% during the final 6h. The protein contents of both the extraembryonic membranes and the embryo were dependent on the glucose uptake.