Bicarbonate-buffered Medium Research Articles

Macromolecules Influence Cellular Competence and Expression Level of IGFs Genes in Bovine Oocytes In Vitro.

Simple SummaryThe macromolecule content of culture media can affect the maturation competence of oocytes, which influences the subsequent in vitro development of embryos. This study was designed to determine the effects of macromolecules on cellular competence and the transcript level of insulin-like growth factors (IGF1, IGF2) and their receptors in bovine oocytes. The current study showed that bovine serum albumin (BSA) and fetal calf serum (FCS) improved nuclear maturation and protein biosynthesis (especially FCS). Polyvinyl alcohol did not support the antioxidant defense mechanism due to decreased glutathione peroxidase enzyme activity. The expression of the IGF1 gene could not be detected in all experimental groups, but BSA and FCS increased the transcript level of the IGF2 gene. Moreover, oocyte maturation with BSA increased the transcript level of the IGF1R gene, whereas the transcript level of the IGF2R gene was similar among macromolecule supplementation groups. The BSA and FCS could improve in vitro bovine oocyte development due to supporting cellular characteristics.In vitro maturation (IVM) of mammalian oocytes, which influences subsequent in vitro development of embryos, is affected by the macromolecule content in culture media for the success of oocyte maturation competence, in which the cytoplasmic and nuclear reprogramming events occur. The insulin-like growth factor family (IGFs) promotes the maturation of bovine oocytes and the expansion of cumulus cells and also inhibits apoptosis. This study was, therefore, designed to examine the effects of macromolecules (bovine serum albumin, BSA; fetal calf serum, FCS; and polyvinyl alcohol, PVA) on in vitro nuclear maturation, total cellular protein, glutathione peroxidase (GPx) enzyme activity, and the gene expression level of IGF1, IGF2, and their receptor in bovine oocytes. Oocytes obtained from bovine ovaries were cultured in bicarbonate-buffered medium 199 supplemented with 4 mg/mL BSA, 10% FCS, 1 mg/mL PVA, and without macromolecule supplement (control) during 22 h in the air with a humidified atmosphere and 5% CO2 at 38.5 °C temperature. Supplementation of BSA and FCS increased (χ2 = 9.84; p < 0.05) the percentages of oocytes that reached metaphase II compared to the control and PVA. The amount of protein per ml of cell extracts of oocytes matured in FCS supplemented culture media was higher (p < 0.05) than the oocytes in the PVA and control. The levels of GPx enzyme activity in cell extracts isolated from oocytes in each experimental group did not change over time, but the GPx enzyme activity in oocytes matured in PVA-supplemented culture media was lower (p < 0.05) than in oocytes in the other experimental groups. Transcript for the IGF1 gene was not detected in all experimental groups, but the supplementation of BSA and FCS significantly elevated the transcript level of the IGF2 gene. In addition, the maturation of oocytes with BSA-supplemented media increased the transcript level of the IGF1R gene, whereas the transcript level of the IGF2R gene was similar among macromolecule supplementation groups. The current study concluded that BSA and FCS could improve in vitro bovine oocyte development due to supporting nuclear maturation and increasing the total cellular protein content, GPx enzyme, and transcript activity.

HEPES-buffering of bicarbonate-containing culture medium perturbs lysosomal glucocerebrosidase activity.

Glucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal GBA allele are at increased risk for Parkinson's disease. GCase undergoes extensive modification of its four N‐glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Fluorescent activity‐based probes (ABPs) that covalently label GCase in reaction‐based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage‐like RAW264.7 cells. Specific attention was paid to the impact of 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) supplementation to bicarbonate‐buffered medium. Here, we report how HEPES‐buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES‐containing medium was also found to reduce maturation of other lysosomal enzymes (α‐glucosidase and β‐glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD‐patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD.

A cell culture platform for quantifying metabolic substrate oxidation in bicarbonate-buffered medium

Complex diseases such as cancer and diabetes are underpinned by changes in metabolism, specifically by which and how nutrients are catabolized. Substrate utilization can be directly examined by measuring a metabolic endpoint rather than an intermediate (such as a metabolite in the tricarboxylic acid cycle). For instance, oxidation of specific substrates can be measured in vitro by incubation of live cultures with substrates containing radiolabeled carbon and measuring radiolabeled carbon dioxide. To increase throughput, we previously developed a miniaturized platform to measure substrate oxidation of both adherent and suspension cells using multiwell plates rather than flasks. This enabled multiple conditions to be examined simultaneously, ideal for drug screens and mechanistic studies. However, like many metabolic assays, this was not compatible with bicarbonate-buffered media, which is susceptible to alkalinization upon exposure to gas containing little carbon dioxide such as air. While other buffers such as HEPES can overcome this problem, bicarbonate has additional biological roles as a metabolic substrate and in modulating hormone signaling. Here, we create a bicarbonate-buffered well-plate platform to measure substrate oxidation. This was achieved by introducing a sealed environment within each well that was equilibrated with carbon dioxide, enabling bicarbonate buffering. As proof of principle, we assessed metabolic flux in cultured adipocytes, demonstrating that bicarbonate-buffered medium increased lipogenesis, glucose oxidation, and sensitivity to insulin in comparison to HEPES-buffered medium. This convenient and high-throughput method facilitates the study and screening of metabolic activity under more physiological conditions to aid biomedical research.

Cooperative Co-Activation of Water and Hypochlorite by a Non-Heme Diiron(III) Complex.

Aqueous solutions of the iron(III) complex of N,N,N'-tris(2-pyridylmethyl)ethylenediamine-N'-acetate (tpena) react with hypochlorite (ClO-) to produce the reactive high-valent [FeIV(O)(tpena)]+. Under catalytic conditions, in bicarbonate-buffered media (pH 8) with a set ionic strength (10 mM NaCl), kinetic analysis shows that two equivalents of [FeIV(O)(tpena)]+ per one ClO- are produced, with benign chloride ions the only byproduct. An unprecedented supramolecular activation of ClO- by {(HCO3)⊂[(tpena)FeIII(μ-O)FeIII(Htpena)]}2+ is proposed. This mode of activation has great advantage for use in the catalytic oxidation of C-H bonds in water since: (i) the catalyst scaffold is protected from oxidative degradation and (ii) undesirable radical side reactions which produce toxic chlorinated compounds are circumvented by this novel coactivation of water and ClO-. The unique activation mechanism by the Fe-tpena system makes possible the destruction of organic contaminants as an add-on technology to water disinfection by chlorination, demonstrated here through (i) the catalytic oxidation of micropollutant metaldehyde, and (ii) mineralization of the model substrate formate. The resting-state speciation at pH 3, 5, 7, and 9, as well as the catalytically active iron speciation are characterized with Mössbauer and EPR spectroscopy and supported by DFT calculations. Our study provides fundamentally new insights into the design and activation mode of iron-based catalysts relevant to applications in water remediation.

Extraction of Au(III) by Microbially Reduced Metal-Organic Frameworks.

Gold is a critical resource in the jewelry and electronics industries and is facing increased consumer demand. Accordingly, methods for its extraction from waste effluents and environmental water sources have been sought to supplement existing mining infrastructure. Redox-mediated treatments, such as Fe(II)-based platforms, offer promise for precipitating soluble Au(III). We hypothesized that microbial generation of Fe(II) in the presence of sorbent metal-organic frameworks could capitalize on the advantages of both biological- and chemical-driven extraction approaches. Toward this aim, we tested Au(III) removal by Shewanella oneidensis cultured with Fe(III)-based materials (ferrihydrite, Fe-BTC, MIL-100, or MIL-127). Across all tested materials, S. oneidensis generated the highest levels of redox-active Fe(II) (1.99 ± 0.27 mM) when cultured with MIL-127 as a respiratory substrate in a bicarbonate-buffered medium. This translated into superior Au(III) removal performance in terms of both removal rate and capacity (k = 2.55 ± 0.60 h-1; Q = 183 mg g-1). Unlike other materials tested, MIL-127 also maintained cell viability following repeated Au(III) challenges, enabling the regeneration of Fe(II) in the framework. Together, these effects facilitated the treatment of multiple cycles of Au(III) by S. oneidensis-reduced MIL-127. Overall, this work demonstrates that microbial generation of Fe(II) can facilitate the removal of Au(III), augmenting purely adsorptive platforms. Given the biological and chemical modularity of our system, our results suggest that future optimizations to microbial Fe(II) generation may offer promise for improving Au(III) extraction processes.

Effective bioreactor pH control using only sparging gases.

pH control is critical in bioreactor operations, typically realized through a two-sided control loop, where CO2 sparging and base addition are used in bicarbonate-buffered media. Though a common approach, base addition could compromise culture performance due to the potential impact from pH excursions and osmolality increase in large-scale bioreactors. In this study, the feasibility of utilizing control of sparge gas composition as part of the pH control loop was assessed in Chinese hamster ovary (CHO) fed-batch cultures. Fine pH control was evaluated in multiple processes at different setpoints in small-scale ambr®250 bioreactors. Desired culture pH setpoints were successfully maintained via air sparge feedback control. As part of the pH control loop, air sparging was increased to improve CO2 removal automatically, hence increase culture pH, and vice versa. The effectiveness of this pH control strategy was seamlessly transferred from ambr®250 to 200 L scale, demonstrating scalability of the proposed methodology. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2743, 2019.

PH: the silent variable significantly impacting meiotic spindle assembly in mouse oocytes.

Temperature fluctuation negatively impacts the assembly and function of the meiotic spindle, but does pH have a similar effect? Polarized light microscopy was used to study the spindle in living mouse oocytes under different pH conditions. Female mice (n = 53) were superovulated, and oocytes collected, denuded and allocated to treatment groups. All experiments were performed at 37°C, and standard bicarbonate-buffered medium was used either pre-equilibrated in 6% CO2 or unequilibrated (in ambient CO2). Mean oocyte spindle retardance was measured over time in response to changing pH. Spindles were also assessed to understand whether this effect was reversible, by using a fixed pH in a zwitterionic buffer. The data show the spindle is impacted by pH fluctuation, with mean retardance significantly higher at pH 7.4-7.5 than at the point of media equilibration (P<0.001). This effect was found to be reversible; retardance significantly decreased after transition of the oocytes from pH 7.43 or pH 7.53 back to the original pre-equilibration pH of 7.32 (P<0.05). This study has shown that the meiotic spindle in mouse oocytes is highly sensitive to changes in oocyte culture media pH. If comparable in humans, this has significance as to the pH level of culture media currently used in assisted reproductive technology clinics worldwide, and reinforces the requirement for stringent control over extrinsic variables in the IVF laboratory.

81 Effects of Day 5 or 6 HEPES-Buffered Transportation Culture Medium on Developmental Competence of Bovine In Vitro-Produced Embryos

Most in vitro-produced (IVP) bovine embryos are transferred fresh. Use of a HEPES/bicarbonate embryo culture medium for transportation would offer flexibility for embryo shipment and transfer. We hypothesized that embryos cultured for 36 (Day 6 embryos) or 60 h (Day 5 embryos) in a novel SCF1T medium (SOF for Conventional Freezing 1 supplemented with HEPES) would maintain developmental competence compared with bicarbonate-buffered medium SCF1 (control). In 5 replicates, IVP embryos were produced by aspirating cumulus–oocyte complexes (COC) from 2-to 8-mm follicles of abattoir ovaries. The COC (n = 1036) were matured for 23 h, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 at 38.5°C, 5% CO2, 5% O2, and 90% N2 (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Randomly, on Day 5 and 6 after fertilization, a subset of presumptive embryos were moved into 500-µL polystyrene vials containing 100 µL of SCF1T medium, covered with 300 µL of sterile mineral oil and cultured in a portable incubator (MicroQ iQ2, Scottsdale, AZ, USA) at 38.5°C for 60 and 36 h, respectively. On Day 7.5 post-fertilization, blastocyst rates were evaluated and embryos (n = 8) from each group were stained with 1 µg mL−1 Nile Red for lipid quantification, and 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial polarity. Images were obtained with confocal microscopy and fluorescent intensity (AFU) was measured by Image J software (National Institutes of Health, Bethesda, MD, USA). Data were analysed by one-way ANOVA and means separated by Tukey’s HSD. Results (Table 1) indicate similar blastocyst rates and lipid content between embryos cultured for 36 to 60 h in SCF1T and control media (P &gt; 0.05). However, mitochondrial polarity was lower in the Day 5 group (P &lt; 0.05) compared with Day 6 and control groups. Results suggest that culturing embryos in SCF1T medium for 36 h maintains developmental competence compared with bicarbonate-buffered media and offers an alternative for shipment and transfer of IVP embryos. Studies involving evaluation of pregnancy rates of the present study are ongoing. Table 1.Effects of Day 5 or 6 SCF1T embryo culture medium on development, lipid content, and mitochondrial polarity

Evaluation of lipid changes in buffalo (Bubalus bubalis) spermatozoa during in-vitro capacitation and acrosome reaction

In this study, buffalo spermatozoa were in-vitro capacitated and acrosome reacted using modified Tyrode’s bicarbonate-buffered medium (sp-TALP media). Lipids were extracted from freshly ejaculated, in-vitro capacitated and acrosome reacted buffalo spermatozoa. The total lipid content of freshly ejaculated spermatozoa (171.5±2.90 μg/108 cells), capacitated spermatozoa (128.2±3.29 μg/108 cells) and acrosome reacted spermatozoa (115.7±2.04 μg/108 cells) were found significantly (P&lt;0.05) different. During the process of capacitation and acrosome reaction (AR) there was substantial loss of total lipids. The total Lipid content of freshly ejaculated, capacitated and acrosome reacted spermatozoa were fractionated into three major classes namely, Phospholipids, Neutral lipids and Glycolipids. Among the classes of lipids; there was no change in concentration of glycolipids. Phospholipids in fresh, capacitated and acrosome reacted spermatozoa were 84.27±1.96, 58.40±2.40 and 50.51±1.27 μg/108 cells respectively. In case of Neutral lipids, the concentration were 33.63±2.62, 22.55±1.86 and 11.61±0.87 μg/108 cells in fresh, capacitated and acrosome reacted spermatozoa respectively. The results generated by this study will be one step further to understand the lipid changes in buffalo spermatozoa during capacitation, acrosome reaction and fertilization process.

Insulinlike growth factor I improves yak (Bos grunniens) spermatozoa motility and the oocyte cleavage rate by modulating the expression of Bax and Bcl-2

The aim of our present study was to examine the effects of insulinlike growth factor 1 (IGF-1) on yak sperm motility during in vitro capacitation and the relationship between the effects of IGF-1 on yak sperm motility and apoptosis was evaluated. Frozen–thawed yak spermatozoa were incubated at 38 °C for 1 hour in Tyrode's bicarbonate-buffered medium for sperm culture (Sp-TALP) with different concentrations (0, 50, 100, and 200 ng/mL) of IGF-1. In every treatment, the sperm motility was measured by a computer-assisted sperm analyzer system. The fertilizing ability of spermatozoa was evaluated on the basis of oocyte cleavage rate after insemination. The expression of Bax and Bcl-2 was examined by real-time polymerase chain reaction and Western blot for the messenger RNA and protein levels. It is interesting to note that IGF-1 improved yak spermatozoa motility and the cleavage rate of oocytes; these improvements were highest in the 100 ng/mL IGF-1 group, followed by the 200 ng/mL and 50 ng/mL groups, with the lowest improvements in motility and cleavage rates in groups without IGF-1. The expression level of Bax was downregulated by IGF-1, whereas Bcl-2 was upregulated. Both messenger RNA and Bax proteins were lowest in groups with 100 ng/mL IGF-1, where the Bcl-2 was the highest. Bax expression in the groups with IGF-1 was lower than that in the group without IGF-1, and Bcl-2 expression was higher in groups with IGF-1 than that in the group without IGF-1. In conclusion, this research reports that improvements in yak spermatozoa motility and the oocyte cleavage rate after the addition of IGF-I may be a result of the reduction of spermatozoa apoptosis rates by modulating the expression of Bax and Bcl-2.

Biological oxidation of Fe(II) in reduced nontronite coupled with nitrate reduction by Pseudogulbenkiania sp. Strain 2002

The importance of microbial nitrate-dependent Fe(II) oxidation to iron biogeochemistry is well recognized. Past research has focused on oxidation of aqueous Fe2+ and structural Fe(II) in oxides, carbonates, and phosphate, but the importance of structural Fe(II) in phyllosilicates in this reaction is only recently studied. However, the effect of clay mineralogy on the rate and the mechanism of the reaction, and subsequent mineralogical end products are still poorly known. The objective of this research was to study the coupled process of microbial oxidation of Fe(II) in clay mineral nontronite (NAu-2), and nitrate reduction by Pseudogulbenkiania species strain 2002, and to determine mineralogical changes associated with this process. Bio-oxidation experiments were conducted using Fe(II) in microbially reduced nontronite as electron donor and nitrate as electron acceptor in bicarbonate-buffered medium under both growth and nongrowth conditions to investigate cell growth on this process. The extents of Fe(II) oxidation and nitrate reduction were measured by wet chemical methods. X-ray diffraction (XRD), scanning and transmission electron microscopy (SEM and TEM), and 57Fe-Mössbauer spectroscopy were used to observe mineralogical changes associated with Fe(III) reduction and Fe(II) oxidation in NAu-2. The bio-oxidation extent under growth and nongrowth conditions reached 67% and 57%, respectively. Over the same time period, nitrate was completely reduced under both conditions to nitrogen gas (N2), via an intermediate product nitrite. Abiotic oxidation by nitrite partly accelerated Fe(II) oxidation rate under the growth condition. The oxidized Fe(III) largely remained in the nontronite structure, but secondary minerals such as vivianite, ferrihydrite, and magnetite formed depending on specific experimental conditions. The results of this study highlight the importance of iron-bearing clay minerals in the global nitrogen cycle with potential applications in nitrate removal in natural environments.

237 CHROMOSOMAL ABNORMALITIES IN IN VITRO-PRODUCED EARLY BOVINE EMBRYOS: USE OF HOMOLOGOUS FOLLICULAR FLUID SUPPLEMENTATION IN THE OOCYTE MATURATION MEDIA

Chromosomal abnormalities were described as a possible cause of embryo failures in cattle, even more so when they are in vitro produced. It has been widely demonstrated that the post-fertilization culture environment affects the frequency of blastomeric aneuploidies. However, the literature concerning the effect of the oocyte maturation techniques on in vitro-produced embryos is scarce. The aim of this study was to determine the effect of homologous bovine follicular fluid (BFF) as a possible replacement for commercial sera in the appearance of chromosomal abnormalities on early IVF-produced embryos. Cumulus–oocyte complexes obtained from ovarian puncture were maturated in modified bicarbonate-buffered TCM-199 media, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin in three different groups. Two treatments were performed: 1) base media supplemented with BFF, obtained aseptically from follicles between 4 and 10 mm in diameter (10 and 20%), and 2) a control group, with base media supplemented with 10% FCS without BFF. After 20 h of culture at 38.5°C in a 5% CO2 humid atmosphere, cumulus–oocyte complexes from both treatments were fertilized in IVF media and then cultured for 72 h in SOF media, according to our laboratory techniques. A total of 152 early embryos were cytogenetically evaluated following our standard laboratory techniques. Developed embryos were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated in each embryo by direct observation at 1250× magnification using a bright field microscope. Results were statistically compared among treatments by the expected proportion test. No significant differences (P &gt; 0.05) were found between different culture media on the percentages of normal diploid embryos or each kind of numerical abnormality. According to our results (Table 1), the use of homologous follicular fluid as a supplement on the oocyte maturation media did not influence the appearance of abnormal complements in the embryos produced compared with the use of FCS. In conclusion, homologous follicular fluid may be considered a valid serum replacement in the maturation media on IVF-produced bovine embryos. Table 1.Analysis of chromosomal complements of Day 3 in vitro-produced bovine embryos derived from oocytes maturated in culture media with different serum supplementation1

Use of γ-hexachlorocyclohexane as a terminal electron acceptor by an anaerobic enrichment culture

The use of γ-hexachlorocyclohexane (HCH) as a terminal electron acceptor via organohalide respiration was demonstrated for the first time with an enrichment culture grown in a sulfate-free HEPES-buffered anaerobic mineral salts medium. The enrichment culture was initially developed with soil and groundwater from an industrial site contaminated with HCH isomers, chlorinated benzenes, and chlorinated ethenes. When hydrogen served as the electron donor, 79–90% of the electron equivalents from hydrogen were used by the enrichment culture for reductive dechlorination of the γ-HCH, which was provided at a saturation concentration of approximately 10mg/L. Benzene and chlorobenzene were the only volatile transformation products detected, accounting for 25% and 75% of the γ-HCH consumed (on a molar basis), respectively. The enrichment culture remained active with only hydrogen as the electron donor and γ-HCH as the electron acceptor through several transfers to fresh mineral salts medium for more than one year. Addition of vancomycin to the culture significantly slowed the rate of γ-HCH dechlorination, suggesting that a Gram-positive organism is responsible for the reduction of γ-HCH. Analysis of the γ-HCH dechlorinating enrichment culture did not detect any known chlororespiring genera, including Dehalobacter. In bicarbonate-buffered medium, reductive dechlorination of γ-HCH was accompanied by significant levels of acetogenesis as well as methanogenesis.

Comparison of the effects of 5% and 6% co2 culture systems for human IVF embryos from woman older than 40 years of age: a prospective randomized study

The negative correlation between fecundity and age in women has been extensively documented. Many efforts have been tried to increase the pregnancy rate in old aged woman(≥40 years of age) undergoing an IVF treatment cycles. While recent development of optimized culture media for culturing human embryos in vitro has provided reliable clinical results, the fertility of aged women remain relatively low. When using bicarbonate-buffered media, the concentration of CO2 influences directly on pH of media and embryo quality.

Effect of natural organic matter on toxicity and reactivity of nano-scale zero-valent iron

Nano-scale zero-valent iron (NZVI) particles are increasingly used to remediate aquifers contaminated with hazardous oxidized pollutants such as trichloroethylene (TCE). However, the high reduction potential of NZVI can result in toxicity to indigenous bacteria and hinder their participation in the cleanup process. Here, we report on the mitigation of the bactericidal activity of NZVI towards gram-negative Escherichia coli and gram-positive Bacillus subtilis in the presence of Suwannee River humic acids (SRHA), which were used as a model for natural organic matter (NOM). B. subtilis was more tolerant to NZVI (1 g/L) than E. coli in aerobic bicarbonate-buffered medium. SRHA (10 mg/L) significantly mitigated toxicity, and survival rates after 4 h exposure increased to similar levels observed for controls not exposed to NZVI. TEM images showed that the surface of NZVI and E. coli was surrounded by a visible floccus. This decreased the zeta potential of NZVI from −30 to −45 mV and apparently exerted electrosteric hindrance to minimize direct contact with bacteria, which mitigated toxicity. H 2 production during anaerobic NZVI corrosion was not significantly hindered by SRHA ( p > 0.05), However, NZVI reactivity towards TCE (20 mg/L), assessed by the first-order dechlorination rate coefficient, decreased by 23%. Overall, these results suggest that the presence of NOM offers a tradeoff for NZVI-based remediation, with higher potential for concurrent or sequential bioremediation at the expense of partially inhibited abiotic reactivity with the target contaminant (TCE).

In Vitro Maturation of Sika Deer (Cervus nippon [Temminck]) Oocytes.

With the ultimate goal of establishing experimental protocols necessary for cloning male Sika deer to gain more pilous antler, the conditions for in vitro maturation of Sika deer oocytes were examined. Sika deer ovaries were collected from Slaughterhouse in oestrus season (October and November), placed into saline (25°C) supplemented with 1% (v/v) penicillin and streptomycin, and transported into the laboratory within 4h. The small vesicular follicles (diameter, 2-6 mm) on the ovarian surface were incised with a scalpel in a Petri dish containing mPBS (Dulbecco PBS supplemented with 0.1% [w/v] D-glucose, 36 mg/L of pyruvate, and 0.4% [w/v] BSA) to release the cumulus-oocyte complexes (COCs). The COCs with uniform cytoplasm and several layers of cumulus cells were selected, divided into black color (more lipid drops) oocytes and white color (little lipid drops) oocytes (more black color oocytes and less white color oocytes were collected), and cultured in two types of media: medium 1: bicarbonate-buffered medium TCM199 with 25 mM NaHCO3, 0.2 mM pyruvate, 0.086 mM kanamycin monosulfate, 10% (v/v) FCS, 10 µg/ml porcine FSH, 1µg/ml bovine LH, 1 µg/ml 17-β-estradiol, and 0.1 mM cysteamine), and medium 2: medium 1 without 17-β-estradiol, for 20, 24, 28 or 48h. In black color oocytes, after 28h of maturation in vitro, the maturation rate (63%, n=43) of cultured in medium 1 was significantly greater (P<0.01) than those of oocytes cultured in vitro for 20h (44%, n=34) and 24h (40%, n=30). And it (63%, n=43) was also higher than that of oocytes cultured in vitro for 48h (53%, n=53), but no significant difference was observed. Same trend was seen in black color oocytes when cultured in medium 2. The maturation rate at 28h (68%, n=37) was higher than those of oocytes cultured in vitro for 20h (53%, n=43), 24h (54%, n=38) and 48h (57%, n=47), but no significant difference was observed among them. In white color oocytes, the maturation rates of oocytes cultured in medium 1 were 17% (n=32) at 28h and 11% (n=38) at 48h. No matured oocytes cultured in vitro for 20h and 24h were seen. Same situation was seen in white color oocytes when cultured in medium 2, the difference was that 6% (n=38) oocytes cultured in vitro for 24h was matured. In this study, we also find that when COCs were cultured in medium 2 (without 17-β-estradiol), their cumulus cells expanded slightly faster and oocytes matured better than those cultured in medium 1. Our results indicate that (i) the best maturation condition of black color oocytes in Sika deer is achieved at 28h by medium 2; (ii) medium 2 (without 17-β-estradiol) is slightly better than medium 1 for in vitro maturation of Sika deer oocytes; (iii) the maturation rate of black color oocytes is much higher than that of white color oocytes when cultured in same medium and same time. These results have begun to elucidate parameters important for animal modeling and cloning with the Sika deer, should facilitate the development of genetically defined animal models in this species.*This work was supported by the National Nature Science Foundation of China (No. 30671510)

A semi‐empirical mathematical model useful for describing the relationship between carbon dioxide, pH, lactate and base in a bicarbonate‐buffered cell‐culture process

The purpose of the present study was to develop a quantitative relationship between the primary factors of state affecting pH control in a bicarbonate-buffered medium. Starting with the Henderson-Hasselbach equation, several assumptions led to the following equation: L = B(T)-s x dCO2 x 10(pH-pK) where L is the lactate concentration (mM), B(T) is the total amount of base added (mM), s is the solubility of CO(2) (mM/%), dCO(2) is the dissolved CO(2) concentration (%) and pK is the acid ionization constant for bicarbonate. This equation appropriately described the relationship of these factors when using bicarbonate, carbonate and HCl (as a lactic acid surrogate) in water. However, the equation required modification to describe the relationship in cell culture medium, due presumably to the presence of other buffers and components; the final form of the equation from an empirical fit in the absence of cells was: L = B(T)-0.88 x dCO2(0.79) x 10(pH-6038) This equation was tested against actual cell culture data, from inoculum preparation in a T-flask through a 10000-litre fed-batch bioreactor, by comparing the lactate concentration calculated from base, pH and dCO(2) data with that actually measured in the bioreactor using a YSI 8500 SELECT Biochemistry Analyzer (YSI Inc., Yellow Springs, OH, U.S.A.). In every case, the calculated and actual lactate concentrations were in good agreement. The equation was useful for isolating the mechanisms leading to varied base addition across 2-, 600- and 10 000-litre-scale bioreactors. This procedure enables a new approach for quantitatively evaluating and understanding factors associated with bioreactor pH control.

Importance of bicarbonate transport for ischaemia‐induced apoptosis of coronary endothelial cells

Bicarbonate transport (BT) has been previously shown to participate in apoptosis induced by various stress factors. However, the precise role of BT in ischaemia-induced apoptosis is still unknown. To investigate this subject, rat coronary endothelial cells (EC) were exposed to simulated ischaemia (glucose free anoxia at Ph 6.4) for 2 hrs and cells undergoing apoptosis were visualized by nuclear staining or by determination of cas-pase- 3 activity. To inhibit BT, EC were either treated with the inhibitor of BT 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS, 300 μmol/l) or exposed to ischaemia in bicarbonate free, 4-(2-hydroxyethyl)-I-piperazi-neethanesulphonic acid (HEPES)-buffered medium. Simulated ischaemia in bicarbonate-buffered medium (Bic) increased caspase-3 activity and the number of apoptotic cell (23.7 + 1.4%versus 5.1 + 1.2% in control). Omission of bicarbonate during ischaemia further significantly increased caspase-3 activity and the number of apoptotic cells (36.7 1.7%). Similar proapoptotic effect was produced by DIDS treatment during ischaemia in Bic, whereas DIDS had no effect when applied in bicarbonate-free, HEPES-buffered medium (Hep). Inhibition of BT was without influence on cytosolic acidification during ischaemia and slightly reduced cytosolic Ca2+ accumulation. Initial characterization of the underlying mechanism leading to apoptosis induced by BT inhibition revealed activation of the mitochondrial pathway of apoptosis, i.e., increase of cytochrome C release, depolarization of mitochondria and translocation of Bax protein to mitochondria. In contrast, no activation of death receptor-dependent pathway (caspase-8 cleavage) and endoplasmic reticulum- dependent pathway (caspase-12 cleavage) was detected. In conclusion, BT plays an important role in ischaemia-induced apoptosis of coronary EC by suppression of mitochondria-dependent apoptotic pathway.

Impaired regulation of pH homeostasis by oxidative stress in rat brain capillary endothelial cells.

(1) Endothelial cells are permanently challenged by altering pH in the blood, and oxidative damage could also influence the intracellular pH (pH(i)) of the endothelium. Cerebral microvascular endothelial cells form the blood-brain barrier (BBB) and pH(i) regulation of brain capillary endothelial cells is important for the maintenance of BBB integrity. The aim of this study was to address the pH regulatory mechanisms and the effect of an acute exposure to hydrogen peroxide (H2O2) on the pH regulation in primary rat brain capillary endothelial (RBCE) cells The RBCE monolayers were loaded with the fluorescent pH indicator BCECF and pH(i) was monitored by detecting the fluorescent changes. (2) The steady-state pH(i) of RBCE cells in HEPES-buffer (6.83 +/- 0.1) did not differ significantly from that found in bicarbonate-buffered medium (6.90 +/- 0.08). Cells were exposed to NH4CI to induce intracellular acidification and then the recovery to resting pH was studied. Half-recovery time after NH4Cl prepulse-induced acid load was significantly less in the bicarbonate-buffered medium than in the HEPES-medium, suggesting that in addition to the Na+ / H+ exchanger, HCO3- / Cl- exchange mechanism is also involved in the restoration of pH(i) after an intracellular acid load in primary RBCE cells. We used RT-PCR-reactions to detect the isoforms of Na+ / H+ exchanger gene family (NHE). NHE-1 -2, -3 and -4 were equally present, and there was no significant difference in the relative abundance of the four transcripts in these cells. (3) No pH(i) recovery was detected when the washout after an intracellular acid load occurred in nominally Na+ -free HEPES-buffered medium or in the presence of 10 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), a specific inhibitor of Na+ / H+ exchanger. The new steady-state pH(i) were 6.37 +/- 0.02 and 6.60 +/- 0.02, respectively. (4) No detectable change was observed in the steady-state pH(i) in the presence of 100 microM H2O2; however, recovery from NH4Cl prepulse-induced intracellular acid load was inhibited when H2O2 was present in 50 or 100 microM concentration in the HEPES-buffered medium during NH4Cl washout. These data suggest that H2O2 is without effect on the activity of Na+ / H+ exchanger at rest, but could inhibit the function of the exchanger after an intracellular acid load.

Metabolic mapping in mammalian retina: a biochemical and 3H-2-deoxyglucose autoradiographic study

It has long been known that mammalian retinas metabolize glucose aerobically to lactic acid and carbon dioxide. The classical view holds that glucose is the primary substrate for energy metabolism in all retinal cells, and that photoreceptor cells have the highest rates of glycolysis and respiration. A different and more recent view is that the Müller cells are the principal, if not sole aerobic producers of lactate, which then serves as the primary fuel for the mitochondria in photoreceptor cells and other retinal neurons. In this paper, we have examined these two competing hypotheses in rat and guinea pig retinas by identifying the cellular sites of glucose uptake and phosphorylation via hexokinase by means of autoradiographic studies with 3H-2-deoxyglucose ( 3H-2DG). The rat retina serves as a vascular model and the guinea pig retina serves as an avascular model. Rat and guinea pig eyecups were incubated in oxygenated, bicarbonate-buffered media containing glucose in the presence of labeled and unlabeled 2DG. Biochemical measurements of lactate production and ATP content were made on rat retinas incubated with different concentrations of glucose and 2DG in order to establish the optimal condition for conducting the autoradiographic studies with 3H-2DG. The optimal substrate concentrations were 1 m m glucose and 0·25 m m 2DG. Results showed that following incubation of dark-adapted rat eyecups for 1 hr in media containing 1 m m glucose/0·25 m m 2DG and supplemented with 3H-2DG, the label was distributed throughout all the layers of the retina, from the ganglion cell layer to the retinal pigment epithelium, with denser label associated with the outer retina (photoreceptors) relative to the density of label in the inner retina, as evaluated by counts of silver grains in individual retinal layers. Exposure of rat eyecups to light did not alter the relative distribution of label, but did increase total grain counts by 70%. However, uptake of labeled 2DG, as measured by scintillation counting of radioactivity in trichloroacetic acid extracts, was not significantly different between light- and dark-adapted rat retinas. In guinea pig eyecups, labeled 2DG was distributed throughout all the retinal layers. Addition of 10 m m lactate or pyruvate to the glucose/2DG media produced no measurable change in the density or distribution of label in the eyecups. Measurements of the activity of hexokinase in rat retinas revealed that this enzyme was present in both the mitochondrial and cytosolic fractions. The present results suggest that as long as the availability of ambient glucose is adequate, retinal neurons use glucose, rather than glial-derived lactate, as the major substrate for the production of high energy phosphates.