High O2 Tensions Research Articles

The Tolerance of Gut Commensal Faecalibacterium to Oxidative Stress Is Strain Dependent and Relies on Detoxifying Enzymes.

Obligate anaerobic bacteria in genus Faecalibacterium are among the most dominant taxa in the colon of healthy individuals and contribute to intestinal homeostasis. A decline in the abundance of this genus is associated with the occurrence of various gastrointestinal disorders, including inflammatory bowel diseases. In the colon, these diseases are accompanied by an imbalance between the generation and elimination of reactive oxygen species (ROS), and oxidative stress is closely linked to disruptions in anaerobiosis. In this work, we explored the impact of oxidative stress on several strains of faecalibacteria. An in silico analysis of complete genomes of faecalibacteria revealed the presence of genes encoding O2- and/or ROS-detoxifying enzymes, including flavodiiron proteins, rubrerythrins, reverse rubrerythrins, superoxide reductases, and alkyl peroxidase. However, the presence and the number of these detoxification systems varied greatly among faecalibacteria. These results were confirmed by O2 stress survival tests, in which we found that strains differed widely in their sensitivity. We showed the protective role of cysteine, which limited the production of extracellular O2•- and improved the survival of Faecalibacterium longum L2-6 under high O2 tension. In the strain F. longum L2-6, we observed that the expression of genes encoding detoxifying enzymes was upregulated in the response to O2 or H2O2 stress but with different patterns of regulation. Based on these results, we propose a first model of the gene regulatory network involved in the response to oxidative stress in F. longum L2-6. IMPORTANCE Commensal bacteria in the genus Faecalibacterium have been proposed for use as next-generation probiotics, but efforts to cultivate and exploit the potential of these strains have been limited by their sensitivity to O2. More broadly, little is known about how commensal and health-associated bacterial species in the human microbiome respond to the oxidative stress that occurs as a result of inflammation in the colon. In this work, we provide insights regarding the genes that encode potential mechanisms of protection against O2 or ROS stress in faecalibacteria, which may facilitate future advances in work with these important bacteria.

Methods to Study NLR in Human Blood Cells.

Autoinflammatory diseases are a group of inherited and multifactorial disorders characterized by an over-activation of innate immune response. In most cases, the clinical manifestations are due to increased activity of the NLRP3 inflammasome resulting in increased IL-1β secretion. Investigating inflammatory cells from subjects affected by autoinflammatory diseases presents a number of technical difficulties related to the rarity of the diseases, to the young age of most patients, to the difficult modulation of gene expression in primary cells. However, since cell stress is involved in the pathophysiology of these diseases, the study of freshly drawn blood monocytes from patients affected by IL-1-mediated diseases strongly increases the chances that the observed phenomena is indeed pertinent to the pathogenesis of the disease and not influenced by the long-term cell culture conditions (e.g., the high O2 tension) or gene transfection in continuous cell lines that may lead to artifacts.

Does Plasma Inhibit the Activity of KCl Cotransport in Red Cells From LK Sheep?

Red cells from LK sheep represent an important paradigm for control of KCl cotransport activity, as well as being important to sheep erythroid function. A previous report (Godart et al., 1997) suggested that autologous plasma markedly inhibits red cell KCC activity and identified the presence of the bicarbonate/CO2 buffer system as the probable cause. Findings were restricted, however, to red cells from patients with sickle cell disease (SCD) swollen anisotonically and carried out at a very high O2 tension (c.700 mmHg). It was therefore important to investigate the generality of the effect described and whether it was also relevant to the two main stimuli for KCC activity encountered most often by circulating red cells in vivo - low pH in active muscle beds during exercise and high urea concentrations in the renal medulla during antidiuresis. Results confirm that inhibition was significant in response to anisotonic swelling with KCC activity in MOPS-buffered saline (MBS) vs. bicarbonate-buffered saline (BBS) and in MBS vs. plasma both reduced (by about 25 and 50%, respectively). By contrast, however, inhibition was absent at low pH and in high concentrations of urea. These findings suggest therefore that red cell KCC activity represents an important membrane permeability in vivo in red cells suspended in plasma. They are relevant, in particular, to sheep red cells, and may also be important by extension to those of other species and to the abnormal red cells found in human patients with SCD.

Canine IVM With SOF Medium, Insulin-Transferrin-Selenium, and Low O2 Tension Improves Oocyte Meiotic Competence and Decreases Reactive Oxygen Species Levels.

Assisted reproductive technologies in canine species are limited due to the low efficiency of in vitro maturation (IVM). Unlike other mammals, bitches ovulate oocytes in the germinal vesicle stage and complete metaphase II (MII) after 48–72 h in the oviductal environment and become fertilizable. For this reason, we compared two different IVM media, synthetic oviductal fluid (SOF) supplemented with 8% bovine serum albumin (BSA) or a mixture of 8% BSA–2.5% fetal bovine serum (FBS) and TCM-199 with 10% FBS. Additionally, we evaluated the effect of supplementation with insulin-transferrin-selenium (ITS) and low O2 tension in oocyte maturation, reactive oxygen species (ROS) levels, membrane integrity, and embryo development following parthenogenetic activation (PA). After 72 h of culture, SOF + BSA, SOF + BSA + FBS, and TCM-199 + FBS show 5, 7, and 4% of MII, respectively, without a statistical difference. However, SOF + BSA produced significantly higher degeneration rates compared to SOF + BSA + FBS (44 and 23%, respectively). Remarkably, supplementation with 1 μl/ml of ITS under high O2 tension demonstrated a beneficial effect by improving maturation rates up to 20% compared to the other groups. Low O2 tension increased maturation rates to 36.5%, although there were no statistical differences compared to high O2 tension in the presence of ITS. Lower ROS levels and higher integrity of the cytoplasmic membrane were found in the presence of ITS despite no differences in maturation rates under low O2 tension groups. Additionally, after PA, 1% development until the eight-cell stage was obtained after activation of in vitro-matured oocytes in the presence of ITS. Taken together, these results indicate that SOF supplemented with 8% BSA and 2.5% FBS is suitable for IVM of canine oocytes and ITS supplementation was beneficial for both high and low O2 tension. Furthermore, the addition of ITS in the cultured system lowers ROS levels and increases membrane integrity in domestic dog oocytes after IVM.

Role of NRF2 in immune modulator expression in developing lung.

After birth, the alveolar epithelium is exposed to environmental pathogens and high O2 tensions. The alveolar type II cells may protect this epithelium through surfactant production. Surfactant protein, SP-A, an immune modulator, is developmentally upregulated in fetal lung with surfactant phospholipid synthesis. Herein, we observed that the redox-regulated transcription factor, NRF2, and co-regulated C/EBPβ and PPARγ, were markedly induced during cAMP-mediated differentiation of cultured human fetal lung (HFL) epithelial cells. This occurred with enhanced expression of immune modulators, SP-A, TDO2, AhR, and NQO1. Like SP-A, cAMP induction of NRF2 was prevented when cells were exposed to hypoxia. NRF2 knockdown inhibited induction of C/EBPβ, PPARγ, and immune modulators. Binding of endogenous NRF2 to promoters of SP-A and other immune modulator genes increased during HFL cell differentiation. In mouse fetal lung (MFL), a developmental increase in Nrf2, SP-A, Tdo2, Ahr, and Nqo1 and decrease in Keap1 occurred from 14.5 to 18.5dpc. Developmental induction of Nrf2 in MFL was associated with increased nuclear localization of NF-κB p65, a decline in p38 MAPK phosphorylation, increase in the MAPK phosphatase, DUSP1, induction of the histone acetylase, CBP, and decline in the histone deacetylase, HDAC4. Thus, together with surfactant production, type II cells protect the alveolar epithelium through increased expression of NRF2 and immune modulators to prevent inflammation and oxidative stress. Our findings further suggest that lung cancer cells have usurped this developmental pathway to promote immune tolerance and enhance survival.

MicroRNA 29 Family Serves a Key Role in Lung Development and the Prevention of Lung Pathology

Even with remarkable advances in the care of preterm infants, chronic lung disease in the form of bronchopulmonary dysplasia (BPD) continues to be a significant pathologic consequence of prematurity. BPD is caused by the interruption of physiologic lung development and exposure of the immature newborn lung to high O2 tensions. BPD is characterized by a simplified alveolar structure, arrested lung growth, impaired vascular development and lung fibrosis (1). To identify effective treatment strategies for BPD, it is important to understand the molecular mechanisms underlying this disorder. MicroRNAs (miRNA, miR) are known to regulate growth, development and repair of the developing lung; whereas, dysregulation of miRNA expression has been associated with pulmonary disease. Specifically, members of the miR-29 family have been linked to pulmonary cancers, fibrosis, and BPD. Previous studies from our laboratory indicate that developmental induction of miR-29 expression in the fetal lung near term serves a key role in promoting surfactant-producing type II cell differentiation and function through repression of TGF-β2 signaling (2). To understand the role of miR-29 in protection against BPD, in the present study, we created mice in which the entire miR-29 family (miR-29a/b1 and miR-29b2/c) (miR-29 dKO) was disrupted. Upon exposure of miR-29 dKO and WT neonatal mice to hyperoxia (95% O2) for 5 days immediately after birth, expression levels of the proinflammatory cytokines and chemokines, IL-1 β, TGF-β1, CXCL2 and IL-6, were significantly increased in the lungs of miR-29 dKO mice, compared to WT. Furthermore, lungs of miR-29 dKO adult mice manifested increased expression of their direct targets, TGF-β2 and TGF-β3. This was associated with increased collagen deposition, as evidenced by enhanced trichrome staining, suggesting the development of lung fibrosis. HDAC4 (a direct target of miR-29) and proinflammatory TNF-α, which have been implicated in pulmonary fibrosis, also were upregulated in the lungs of miR-29 dKO mice. Overall, our studies suggest a key role of the miR-29 family and its targets in prevention of inflammatory and profibrotic signaling in the neonatal lung leading to lung pathology.Supported by: NIH R01-HL050022 (C.R.M.)

Red blood cell carbonic anhydrase mediates oxygen delivery via the Root effect in red drum.

Oxygen (O2) and carbon dioxide (CO2) transport are tightly coupled in many fishes as a result of the presence of Root effect hemoglobins (Hb), whereby reduced pH reduces O2 binding even at high O2 tensions. Red blood cell carbonic anhydrase (RBC CA) activity limits the rate of intracellular acidification, yet its role in O2 delivery has been downplayed. We developed an in vitro assay to manipulate RBC CA activity while measuring Hb-O2 offloading following a physiologically relevant CO2-induced acidification. RBC CA activity in red drum (Sciaenops ocellatus) was inhibited with ethoxzolamide by 53.7±0.5%, which prompted a significant reduction in O2 offloading rate by 54.3±5.4% (P=0.0206, two-tailed paired t-test; n=7). Conversely, a 2.03-fold increase in RBC CA activity prompted a 2.14-fold increase in O2 offloading rate (P<0.001, two-tailed paired t-test; n=8). This approximately 1:1 relationship between RBC CA activity and Hb-O2 offloading rate coincided with a similar allometric scaling exponent for RBC CA activity and maximum metabolic rate. Together, our data suggest that RBC CA is rate limiting for O2 delivery in red drum.

3094 – ROLE OF BONE MARROW INJURY IN PROMOTING TET2-MEDIATED CLONAL HEMATOPOIESIS OF INDETERMINATE POTENTIAL (CHIP)

CHIP is characterized by selective outgrowth of hematopoietic stem/progenitor cell (HSPC) in the bone marrow (BM) which frequently harbors oncogenic mutations in genes such as TET2. This leads to aberrant expansion of hyperinflammatory immune cells that can contribute to cardiovascular disease and other comorbidities. Prior genotoxic treatments such as chemotherapy and radiation reportedly correlate with the expansion of CHIP mutant HSPC. However, the mechanism(s) remain poorly understood. To address this question, we modeled human CHIP via adoptively transferring Tet2-/- mouse BM into wild-type (WT) recipient mice. In this setting, Tet2-/- HSPC and their progeny preferentially expanded in the BM and blood. Interestingly, Tet2-/- HSPC also colonized non-hematopoietic organs including lung, liver and gonadal adipose tissue. To test whether genotoxic stress caused by radiation accelerated this process, we subjected Tet2-/- CHIP mice to 2.5Gy radiation. Indeed, radiation accelerated Tet2-/- HSPC expansion in nearly all tissues. To assess the impact of microenvironmental factors induced by radiation on CHIP progression, we modeled radiation-induced inflammation and tissue oxygenation using in vitro HSPC culture. Surprisingly, inflammatory cytokines were not sufficient to drive selective expansion of Tet2-/- HSPC. On the other hand, Tet2-/- HSPC displayed a significant proliferative advantage over WT when cultured at high oxygen tension (20%O2) but not in hypoxic conditions representative of healthy BM (1%O2), suggesting high O2 tension caused by BM injury may play a vital role in the selective expansion of Tet2-/- HSPC. Ongoing studies are aimed at understanding the impact of increased oxygen levels in the BM using non-genotoxic approaches and identifying mechanism(s) by which Tet2-/- HSPC harness high O2 tension to proliferate. Such investigations could provide a basis for preventing CHIP and its associated comorbidities.

35 Ethanolic extracts of Cerrado plants in cryotolerance of invitro-produced bovine embryos

Invitro embryo culture induces excessive production of reactive oxygen species (ROS) that impair the quality of produced blastocysts, making them less cryotolerant. Supplementation of culture media with antioxidants would be an alternative to minimise these effects. The present study evaluated the effect of ethanolic extracts obtained from Brazilian Cerrado plants on the cryotolerance of invitro-produced embryos. Bovine ovaries from a slaughterhouse were used to obtain grade I and II oocytes, which were submitted to maturation, fertilization (Day 0), and invitro culture. After fertilization, zygotes were distributed into five groups: one fresh control group (GCont), cultured in high O2 tension (~21%), and four groups cryopreserved at Day 7: a control group subject to high O2 tension without extract supplementation (GDT); a group (G5%) cultured in low O2 tension (5%) without extract supplementation, and two groups cultured under high O2 tension, one supplemented with 0.01mgmL−1 of the ethanolic cagaita extract (Eugenia dysenterica; GCag) and another with 0.01mgmL−1 of murici extract (Byrsonima crassifolia; GMur). Only the expanded blastocysts (EB) of each group were submitted to the direct-transfer cryopreservation system, with slow freezing with ethylene glycol (Sanches et al. 2016 Theriogenology 85, 1147-1151). A total of 163 thawed embryos were evaluated 12, 24, and 36h post-thawing for re-expansion, hatching, and embryonic degeneration rate. In addition, after 12h of post-thaw culture, only the EB embryos were subjected to ROS measurement, quantified in confocal microscopy using 2′,7′-dichlorodihydrofluorescein diacetate, and the rate of apoptosis was obtained by the terminal deoxynucleotidyl transferase dUTP nick end labelling method. Data were analysed by analysis of variance, and the means were compared by TUKEY. A total of 133 EB were used (GCont: 20; GDT: 22; GCag: 35; GMur: 36; G5%: 20). Results are expressed as means±standard deviation. No differences were observed on re-expansion among treatments and time of culture. However, 24h post-thaw, hatching and degeneration rates differed between groups. The group GCont had a higher (P&amp;lt;0.05) hatching rate (47.5±10.3) than the GDT (14.4±6.7) and G5% (10.5±5.8) groups but was similar (P&amp;gt;0.05) to groups supplemented with extracts GCag (22.2±17.1) and GMur (17.5±17.5). Degeneration rate was higher (P&amp;lt;0.05) at 12h for the direct-transfer group (30.2±11.3) and similar among the others (GCont: 0; GCag: 16.8±12; GMur: 10.8±10.8; G5%: 11±11). Nevertheless, GCag had lower embryonic degeneration rates (20±16.7) than the others cryopreserved groups 24h after thawing (GDT: 37.7±5.5; GMur: 38.9±8.2; G5%: 41.3±14.4). Although ROS levels were similar among all groups (P&amp;gt;0.05), apoptosis rate was higher (P&amp;lt;0.05) in the GDT group (23.4±1.9) than in the GCont groups (10.6±1.3): GCag (10.8±1.1), GMur (10.1±1.1), and G5% (7.8±1.2). Therefore, supplementation of ethanolic extracts (0.01mgmL−1) of cagaita and murici improved the embryo quality by reducing the degeneration and apoptosis rates of cryopreserved embryos when compared with the group without supplementation (GDT). Such extracts may be an alternative to increase the cryotolerance of invitro-produced bovine embryos.

The quaternary state of polymerized human hemoglobin regulates oxygenation of breast cancer solid tumors: A theoretical and experimental study.

A major constraint in the treatment of cancer is inadequate oxygenation of the tumor mass, which can reduce chemotherapeutic efficacy. We hypothesize that polymerized human hemoglobin (PolyhHb) can be transfused into the systemic circulation to increase solid tumor oxygenation, and improve chemotherapeutic outcomes. By locking PolyhHb in the relaxed (R) quaternary state, oxygen (O2) offloading at low O2 tensions (<20 mm Hg) may be increased, while O2 offloading at high O2 tensions (>20 mm Hg) is facilitated with tense (T) state PolyhHb. Therefore, R-state PolyhHb may deliver significantly more O2 to hypoxic tissues. Biophysical parameters of T and R-state PolyhHb were used to populate a modified Krogh tissue cylinder model to assess O2 transport in a tumor. In general, we found that increasing the volume of transfused PolyhHb decreased the apparent viscosity of blood in the arteriole. In addition, we found that PolyhHb transfusion decreased the wall shear stress at large arteriole diameters (>20 μm), but increased wall shear stress for small arteriole diameters (<10 μm). Therefore, transfusion of PolyhHb may lead to elevated O2 delivery at low pO2. In addition, transfusion of R-state PolyhHb may be more effective than T-state PolyhHb for O2 delivery at similar transfusion volumes. Reduction in the apparent viscosity resulting from PolyhHb transfusion may result in significant changes in flow distributions throughout the tumor microcirculatory network. The difference in wall shear stress implies that PolyhHb may have a more significant effect in capillary beds through mechano-transduction. Periodic top-load transfusions of PolyhHb into mice bearing breast tumors confirmed the oxygenation potential of both PolyhHbs via reduced hypoxic volume, vascular density, tumor growth, and increased expression of hypoxia inducible genes. Tissue section analysis demonstrated primary PolyhHb clearance occurred in the liver and spleen indicating a minimal risk for renal damage.

134 Effects of Gas Tension During Culture upon Development of Bovine Embryos from Beef (Nellore) and Dairy (Girolando) Breeds

Culture of bovine embryos is a critical step during in vitro embryo production (IVEP) and, as such, has been the focus of numerous studies on cattle IVEP. Improvements of culture conditions to mimic the in vivo maternal microenvironment involves studying the optimal gas tension for pre-implantation embryonic development. In the commercial conditions, there is great variability in results, in part because of the difference between breeds and donors. The objective of this study was to evaluate the effects of culture in high or low oxygen tension upon the development of embryos from a crossbred dairy breed (Girolando F1; Gir × Holstein) and a beef Bos indicus breed, Nellore. We collected data from an IVEP commercial operation located in a tropical area of southeastern Brazil (Minas Gerais State) from February to May 2017. The study was designed in a 2 × 2 factorial arrangement of treatments: 2 O2 tensions during culture (5%, low O2 v. 20%, high O2) and 2 breeds (Nellore, beef v. Girolando F1, dairy). Thus, the following 4 groups were studied: Nellore-high O2 (n = 86 donors), Nellore-low O2 (n = 107 donors), Girolando F1-high O2 (n = 114 donors), and Girolando F1-low O2 (n = 110 donors). Outcome variables were the number of cleaved embryos 72 h post-insemination (hpi), cleavage rate relative to the total number cumulus–oocyte complexes (COC) put in culture, number and percentage of blastocysts 192 hpi relative to the structures kept in culture. Variables that were not normally distributed were transformed using the formula log(y + 0.05). Data were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA) for the main effects of gas tension (low v. high O2) and breed (Girolando F1 v. Nelore). Results are shown as mean ± SEM. Gas tension affected the number of cleaved embryos (10.52 ± 0.92 v. 8.33 ± 0.72 for high and low O2, respectively; P &lt; 0.01) and cleavage rates (40.58 ± 2.49 v. 44.41 ± 2.88 for high and low O2; P &lt; 0.01 in Nellore), but did not affect these variables in Girolando F1 donors (13.23 ± 1.33 v. 10.76 ± 0.76 cleaved embryos, for high and low O2; P = 0.63; 58.01 ± 2.00 v. 60.19 ± 1.97 cleavage rate, for high and low O2; P = 0.80). Nonetheless, the number and percentage of blastocysts were not affected by gas tension in either breed. Results for Nellore were 4.99 ± 0.56 v. 3.51 ± 0.38 blastocysts in high and low O2, respectively (P = 0.051) and 41.92 ± 3.91% v. 39.81 ± 3.77% blastocysts, in high and low O2 (P = 0.11). For Girolando F1, numbers of blastocysts were 5.84 ± 0.66 v. 4.24 ± 0.39 in high and low O2 (P = 0.19) and percentage of blastocysts 49.14 ± 2.97% v. 49.11 ± 3.40% in high and low O2 (P = 0.46). These results suggest that oxygen tension during culture affects IVEP differently depending on breed. The initial period of culture, recognised as critical in IVEP, seemed more sensitive to high O2 tension, particularly in Nellore.

NLR in Human Diseases: Role and Laboratory Findings.

Autoinflammatory diseases are a group of inherited and multifactorial disorders characterized by an overactivation of innate immune response. In most cases, the clinical manifestations are due to increased activity of the NLRP3 inflammasome resulting in increased IL-1β secretion. Investigating inflammatory cells from subjects affected by autoinflammatory diseases presents a number of technical difficulties related to the rarity of the diseases, to the young age of most patients, and to the difficult modulation of gene expression in primary cells. However, since cell stress is involved in the pathophysiology of these diseases, the study of freshly drawn blood monocytes from patients affected by IL-1-mediated diseases strongly increases the chances that the observed phenomena is indeed pertinent to the pathogenesis of the disease and not influenced by the long-term cell culture conditions (e.g., the high O2 tension) or gene transfection in continuous cell lines that may lead to artifacts.

Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli

Abstract H2 production under aerobic conditions has been proposed as an alternative method to overcome the fundamentally low yield of H2 production by fermentative bacteria by maximizing the number of electrons that are available for H2. Here, we engineered Vitreoscilla hemoglobin (VHb) in Escherichia coli to study the effects of this versatile oxygen (O2)-binding protein on oxic H2 production in a closed batch system that was supplemented with glucose. The H2 yields that were obtained with the VHb-expressing E. coli were greatly enhanced in comparison to the negative control cells in culture that started with high O2 tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing cells was also much higher than that of the negative control cells. Through inhibitor studies and time-course experiments, VHb was shown to contribute to the improved H2 yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase before the onset of H2 production and not by working as an O2-scavenger during H2 production. This new approach allowed more substrate to remain to be further utilized for the production of more H2 from limited resources. We expect that VHb can be successfully engineered in potential aerobic H2-producing microbial systems to enhance the overall H2 production yield. In addition, the remarkably high FHL activity of oxically grown, VHb-expressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H2.

Environmental Determinants of Transformation Efficiency in Helicobacter pylori

Helicobacter pylori uses natural competence and homologous recombination to adapt to the dynamic environment of the stomach mucosa and maintain chronic colonization. Although H. pylori competence is constitutive, its rate of transformation is variable, and little is known about factors that influence it. To examine this, we first determined the transformation efficiency of H. pylori strains under low O2 (5% O2, 7.6% CO2, 7.6% H2) and high O2 (15% O2, 2.9% CO2, 2.9% H2) conditions using DNA containing an antibiotic resistance marker. H. pylori transformation efficiency was 6- to 32-fold greater under high O2 tension, which was robust across different H. pylori strains, genetic loci, and bacterial growth phases. Since changing the O2 concentration for these initial experiments also changed the concentrations of CO2 and H2, transformations were repeated under conditions where O2, CO2, and H2 were each varied individually. The results showed that the increase in transformation efficiency under high O2 was largely due to a decrease in CO2. An increase in pH similar to that caused by low CO2 was also sufficient to increase transformation efficiency. These results have implications for the physiology of H. pylori in the gastric environment, and they provide optimized conditions for the laboratory construction of H. pylori mutants using natural transformation.

Reduced oxygen tension improves embryo quality but not clinical pregnancy rates: a randomized clinical study into ovum donation cycles

To investigate the effect of low O2 tension during in vitro culture in terms of ongoing pregnancy rates in ovum donation cycles. Randomized trial. Private university-affiliated IVF center, university-based hospital. A total of 1,125 cycles of ovum donation. Embryo culture in an atmosphere of 5.5% CO2, 6% O2, and 88.5% N2 versus a dual-gas system of 5.5% CO2 in air. Ongoing clinical pregnancy rates per intention-to-treat (ITT) patients. The use of low O2 tension achieved a 41.3% ongoing pregnancy rate per ITT compared with a 40.8% rate obtained for 5% CO2 in air. The mean number of blastomeres and the percentage of top-quality embryos were significantly higher after lower O2 concentration during in vitro culture (7.1 ± 3.6 and 28.6% vs. 7.3 ± 8.4 and 32.1%, respectively). In the ovum donation cycles undergoing day-3 embryo transfers, the use of low O2 tension did not improve ongoing pregnancy rates per cycle and per transfer. However, it benefited embryo quality, demonstrating the potential negative impact of high O2 tension on the in vitro embryo development.

In Vitro Propagation of Mesenchymal Stromal Cells From Marrow Aspirates of Patients with Chronic Lymphocytic Leukemia Is Dependent Upon Physiologic Oxygen Tension

Abstract 2839The tumor microenvironment may play an important role in the growth and/or survival of leukemia cells of patients (pts) with chronic lymphocytic leukemia (CLL). Studies on the interaction of CLL cells with the microenvironment have been facilitated by our capacity to culture accessory cells in vitro. However, the conditions for culturing such cells in ambient oxygen(O2) at 21% are different than those present in lymphoid tissues which have O2 concentrations ranging from 1–7%. The difference between in vivo and in vitro O2 tensions might influence the biology of leukemia accessory cells.To examine this, we studied the effect(s) of O2 tension on our ability to propagate mesenchymal stromal cells (MSCs) from marrow aspirates of pts with CLL. Equal numbers of fresh or viably frozen marrow mononuclear cells were seeded in DMEM with 10% FBS into separate flasks for culture at 37° C in incubators at atmospheric O2 (Atmos-O2) or at 5% O2 (physiologic; Phys-O2), both with 5% CO2. The cells were monitored for viability and growth over time. We found that only Phys-O2 tension allowed for the generation and long-term expansion of MSCs. Out of the 6 pts tested, 3 developed virtually no MSCs (<10 cells), and 3 generated less than 6×104 MSCs (ranging from 2±1×104 to 6±1×104) in Atmos-O2 after 47±7 days in vitro. In contrast, high numbers of MSCs developed in Phys-O2 for all 6 pts, ranging from 45±8×104 to 80×104 cells, resulting in highly significant differences in yields between the 2 culture conditions (p<0.01). Moreover, the MSCs generated in Phys-O2 continued to proliferate over time, whereas MSCs in Atmos-O2 did not. Under Phys-O2, MSCs were successfully expanded from marrow aspirates of 16 out of 18 CLL pts. The morphology and phenotype of the MSCs generated were similar to that of healthy MSCs, expressing CD29, CD44, CD105 and D7-FIB, and lacking expression of CD14, CD31, CD34, or CD45.We next examined whether the differences in cell yields between the two culture conditions could be the result of compromised MSC proliferation in Atmos-O2. To address this, MSCs generated in Phys-O2 were seeded into separate flasks and exposed to Atmos-O2 or Phys-O2 and proliferation was monitored by BrdU incorporation and viable cell counts. We found that MSCs seeded in Atmos-O2 proliferated significantly less well than MSCs in Phys-O2 (n=3). However, MSC viability was not significantly affected by the change in O2 tension, suggesting that replicative senescence could be induced in MSCs exposed to Atmos-O2. To test this hypothesis, MSCs generated in Phys-O2 were seeded separately under Atmos-O2 or Phys-O2 and stained for the senescence-associated beta-galactosidase (SA-B-Gal) marker. We found a significant increase in the fraction SA-B-gal+ MSCs exposed to Atmos-O2 compared to Phys-O2 (70±18% vs. 13±5%; p<0.0001). MSC morphology in Atmos-O2 was also consistent with senescence, marked by a wide-spread cytoplasm and enlarged nucleus. The cell cycle regulator p16INK4 also was distinctively induced in MSCs exposed to Atmos-O2 compared to Phys-O2 (n=2), consistent with its role in inhibiting cell cycle progression and mediating senescence.We next ask if a disruption of the redox balance plays a role in MSC biology modulated by O2, using the free radical scavenger beta-mercaptoethanol (BME). MSCs generated in Phys-O2 were seeded separately under Phys-O2 or Atmos-O2 +/− BME. We observed that MSC proliferation in Atmos-O2 was restored by the addition of BME to the levels observed in Phys- O2 using BrdU incorporation (n=3). However, in a similar context, BME did not restore MSC expansion in Atmos-O2 measured by viable cell counts, or in the generation of MSCs in Atmos-O2 when added at culture initiation (n=2), suggesting that redox balance disruption is not the main mechanism by which high O2 tension affects MSC biology.We finally evaluated whether co-cultures of CLL cells and MSCs in Phys-O2 and Atmos-O2 tensions equally support CLL cell survival. We found that CLL cell survival was significantly enhanced when co-cultured in Phys-O2 compared to Atmos-O2 after 17 days (87±15% vs 44±17% viable cells; p<0.0001). These results suggest that Phys-O2 tension is not only critical to generate MSCs in vitro, but it also has a profound impact on the biology of these accessory cells, which in turn affects the survival of the leukemic cells. Studies conducted under Phys-O2 tension might further our understanding of the mechanisms governing CLL cell survival in vivo. Disclosures:No relevant conflicts of interest to declare.

Embryo development and pregnancy rates of in vitro produced bovine embryos cultured in high and low-oxygen (O 2) atmosphere tension with sequential media (SM)

OBJECTIVE: To evaluate embryo development and pregnancy rates with two O2 concentrations in bovine embryo culture, using SM.DESIGN: Randomized trial.MATERIALS AND METHODS: Oocytes collected from slaughterhouse ovaries cows were matured and fertilized in vitro using standard procedures. The resultants zygotes were cultured in a media 1 plus bovine serum albumin-fatty acid free (3 mg/ml) for 96 hours post-insemination (hpi) followed by a media 2 for 48 hours additional culture (Koyner et al., 2010), at 38.5°C in either an atmosphere of 6% CO2 and 20% O2 (treatment 1; n= 500) or an atmosphere of 6% CO2, 5%O2 and 89% N2 (treatment 2; n= 550). A subset of quality grade 1 blastocysts produced in treatment 1 (n= 25) or treatment 2 (n= 25) were transferred nonsurgically to synchronized recipients. The experiment was replicated four times and data were analyzed using chi-square.Table 1Effect of O2 tension during bovine embryo culture with SM.TreatmentZygotes (n)≥2 cells; 48hpi (%)≥8 cells; 96 hpi (%)Blastocysts; 144 hpi (%)1500420 (84%)272 (54%)191 (38%)2550468 (85%)330 (60%)242 (44%)No significant differences. Open table in a new tab CONCLUSION: These results demonstrate that SM employed in this study, which contain antioxidants and growth factors, can support in vitro embryo development and his viability under atmosphere of high O2 tension in a similar way to atmosphere of low O2 tension. Additional transfers are needed to determine if the use of this SM will avoid applying low O2 tension during bovine culture, which could be important regarding high costs of this system. OBJECTIVE: To evaluate embryo development and pregnancy rates with two O2 concentrations in bovine embryo culture, using SM. DESIGN: Randomized trial. MATERIALS AND METHODS: Oocytes collected from slaughterhouse ovaries cows were matured and fertilized in vitro using standard procedures. The resultants zygotes were cultured in a media 1 plus bovine serum albumin-fatty acid free (3 mg/ml) for 96 hours post-insemination (hpi) followed by a media 2 for 48 hours additional culture (Koyner et al., 2010), at 38.5°C in either an atmosphere of 6% CO2 and 20% O2 (treatment 1; n= 500) or an atmosphere of 6% CO2, 5%O2 and 89% N2 (treatment 2; n= 550). A subset of quality grade 1 blastocysts produced in treatment 1 (n= 25) or treatment 2 (n= 25) were transferred nonsurgically to synchronized recipients. The experiment was replicated four times and data were analyzed using chi-square. No significant differences. CONCLUSION: These results demonstrate that SM employed in this study, which contain antioxidants and growth factors, can support in vitro embryo development and his viability under atmosphere of high O2 tension in a similar way to atmosphere of low O2 tension. Additional transfers are needed to determine if the use of this SM will avoid applying low O2 tension during bovine culture, which could be important regarding high costs of this system.

253 DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES MATURED IN SERUM-FREE MEDIUM UNDER LOW OXYGEN TENSION

In vitro maturation (IVM) is a critical step in in vitro bovine embryo production. A number of factors can influence the IVM environment, such as media composition and protein supplementation. Serum and higher O2 tension have been shown to reduce embryo quality; however, little is known about the effects of serum and O2 tension during IVM on embryo quality and development. This study aimed to evaluate the effect of serum and O2 tension on IVM of bovine oocytes. Immature oocytes obtained from slaughterhouse ovaries were randomly distributed in 4 groups of IVM: G1 (n = 253), 0.1% polyvinyl alcohol (PVA) in air; G2 (n = 248), 10% inactivated estrous cow serum (ECS) in air; G3 (n = 270), 0.1% PVA under 5% O2; and G4 (n = 236), 10% ECS under 5% O2. In vitro maturation was performed with TCM-199 culture medium supplemented with 20 μg mL–1 FSH, under 5% CO2 at 38.5°C for 24 h. After maturation, oocytes were in vitro fertilized with 2.0 × 106 sperm mL–1 in Fert TALP medium, supplemented with heparin, for 20 h. Presumptive zygotes were denuded by vortexing and cultured in CR2aa medium with 2.5% fetal calf serum under 5% CO2 and 5% O2 at 38.5°C. Cleavage rate was evaluated 72 h postfertilization, and blastocyst rate and total cell number were evaluated 8 days postfertilization. Morphological classification of embryos was performed at Day 8 according to the International Embryo Transfer Society manual (1998). Cleavage, blastocyst, and grade 1 embryo rates were analyzed by chi-square, and total cell number was analyzed by ANOVA, with means compared by LSD. Results are presented as mean ± SEM. There was no difference (P &gt; 0.05) in cleavage rates among G1, G2, and G4 (61.6, 65.3, and 57.6%, respectively), but cleavage rate was lower (P &lt; 0.05) in G3 (52.5%). Blastocyst rates among G1, G3, and G4 (15.8, 17.7, and 20.3%, respectively) were similar (P &gt; 0.05). However, blastocyst rate in G2 (25.0%) was higher (P &lt; 0.05) than in G1 and G3, but was similar to G4 (P &gt; 0.05). Total cell number was similar (P &gt; 0.05) among G2 (194.1 ± 13.1), G3 (173.3 ± 9.0), and G4 (163.8 ± 8.7), but was lower (P &lt; 0.05) in G1 (124.5 ± 11.4). The grade 1 embryo rate was lower (P &lt; 0.05) in G1 (70.3%) than in G2 (89.5%), but was similar (P &gt; 0.05) to G3 (77.0%) and G4 (83.9%). The results suggest that IVM with PVA in TCM-199 medium under 5% O2 can be performed without reducing embryo development and quality, when compared with ECS. On the other hand, oocyte developmental competence seems to be affected when IVM is performed with PVA under air conditions. Financial support: CNPq, FAPEMIG.

247 EFFECTS OF CYSTEINE IN IVM MEDIA ON IN VITRO MATURATION UNDER LOW OXYGEN TENSION, IN VITRO FERTILIZATION, AND IN VITRO CULTURE OF PORCINE OOCYTES

Cysteine is considered to promote male pronuclear (MPN) formation in porcine through oocyte glutathione (GSH) synthesis (Yoshida et al. 1993 Biol. Reprod. 49, 89–94). The GSH has an important role in providing cells with a redox state and in acting to protect cells from toxic effects of oxidative damage (Meister et al. 1976 AM Rev. Biochem. 45, 559–604). However, such previous investigations were carried out under high O2 tension (20% O2) incubation conditions. Here we simply study IVM-IVF-IVC competence of porcine oocytes matured in IVM media supplemented with cysteine of different concentrations under low oxygen tension (5% O2). Cumulus–oocyte complexes (COCs) from prepubertal gilts were collected, matured, and fertilized in vitro according to Kikuchi et al. (2000 Biol. Reprod. 66, 1033–1041). COCs were cultured in IVM medium supplemented with 0 (Group 1; control), 0.05 (Group 2), 0.1 (Group 3), 0.2 (Group 4), and 0.6 mm (Group 5) cysteine under low oxygen tension. Nuclear maturation of oocytes, fertilization status, and number of cells in resultant embryos were assessed with orcein staining; also, the GSH content of IVM oocytes was measured by the method described by Ozawa et al. (2002 Reproduction 124, 683–689). Maturation rates of Groups 1–5 were 68.2 � 3.2, 70.6 � 7.7, 69.7 � 15.9, 75.9 � 7.7, and 68.8 � 8.0%, respectively, indicating no difference in maturation competence among the groups (P &gt; 0.05 by ANOVA). The rates of sperm penetration, MPN formation (95.9 � 2.4, 100 � 0, 92.8 � 4.7, 94.0 � 4.1, and 92.4 � 2.7%, respectively), monospermy, and even blastocyst rates after 6 days of IVC were not different among the groups (P &gt; 0.05 by ANOVA). Moreover, the cell numbers of blastomeres in blastocysts (38.68 � 3.5, 40.1 � 3.1, 37.5 � 3.0, 36.2 � 3.3, and 43.8 � 4.0, respectively) were uniformly the same among the groups (P &gt; 0.05 by ANOVA). However, GSH content of IVM oocytes increased significantly (P &lt; 0.05 by ANOVA) as the concentration of cysteine increased (12.2 � 0.6, 14 � 0.8, 15.1 � 0.5, 16.4 � 0.4, and 16.4 � 0.5 pmol/oocyte, respectively). The GSH level of oocytes in Group 1 (control) seems to be higher than that reported by Aberydeera et al. (1998 Biol. Reprod. 58, 213–218), who matured porcine oocytes under high O2 tension. This may reflect the effect of low O2 tension and explain the same developmental rate to the blastocyst stage as that of oocytes matured in the media supplemented with cysteine in this study. In conclusion, an addition of 0.05–0.6 mm cysteine during IVM, under 5% O2 tension, of porcine oocytes significantly increased intracellular GSH synthesis according to its concentration. However, it had no promoting effects on nuclear maturation, fertilization, male pronucleus formation, and subsequent embryonic development to the blastocyst stage. Thus, O2 tension during IVM of oocytes is suggested to be important for the in vitro production of porcine blastocysts.

Oxygen tension modulates the expression of pulmonary vascular BKCachannel α- and β-subunits

At birth, the lung environment changes from low to relatively high O(2) tension. Pulmonary blood flow increases and pulmonary artery pressure decreases. Recent data suggest that pulmonary vascular calcium-sensitive K(+) channel (BK(Ca)) activation mediates perinatal pulmonary vasodilation. Although BK(Ca) channel expression is developmentally regulated, the molecular mechanisms responsible for BK(Ca) expression remain unknown. We tested the hypothesis that the low-O(2) tension environment of the normal fetus modulates BK(Ca) channel expression. We analyzed BK(Ca) expression under conditions of hypoxia and normoxia both in vitro and in vivo. BK(Ca) alpha-subunit mRNA expression increased twofold in ovine pulmonary artery smooth muscle cell (PASMC) primary cultures maintained in hypoxia. In vivo, BK(Ca) expression was similarly affected by hypoxia. When adult Sprague-Dawley rats were placed in hypobaric hypoxic chambers for 3 wk, hypoxic animals showed an increase of threefold in the expression of BK(Ca) alpha- and more than twofold in the expression of BK(Ca) beta(1)-subunit mRNA. Immunochemical staining was consistent with the genetic data. To assess transcriptional activation of the beta-subunit of the BK(Ca), both BK(Ca) beta(1)- and beta(2)-subunit luciferase (K(Ca) beta:luc(+)) reporter genes were constructed. Hypoxia increased PASMC K(Ca) beta(1):luc(+) reporter expression by threefold and K(Ca) beta(2):luc(+) expression by 35%. Fetal PASMC treated with the hypoxia-inducible factor-1 mimetic deferoxamine showed a 63 and 41% increase in BK(Ca) channel alpha- and beta(1)-subunit expression, respectively. Together, these results suggest that oxygen tension modulates BK(Ca) channel subunit mRNA expression, and the regulation is, at least in part, at the transcriptional level.