The relationship of oxidative stress, adiposity and metabolic risk factors in healthy Black and White American youth.

UNLABELLED: What is already known about this subject African Americans are disproportionately affected by obesity and other metabolic risk factors in comparison to White Americans. Increasing prevalence of obesity has been associated with concomitant increases in childhood hypertension, dyslipidaemia and type 2 diabetes. Oxidative stress is associated with obesity in both adults and children. What this study adds Oxidative stress is positively associated with total body fat and truncal fat, but not with body mass index (BMI) or BMI z-score in healthy youth. Oxidative stress is associated with diastolic blood pressure in African American but not in White American healthy youth. BACKGROUND: Oxidative stress is elevated in obese youth, but less is known regarding racial disparities in the relationship of oxidative stress with metabolic risk factors. OBJECTIVES: To determine the relationship between oxidative stress and metabolic risk factors, adiposity, leptin, adiponectin and cardiovascular fitness (VO2PEAK ) in healthy African American and White American youth. METHODS: A marker of oxidative stress (F2 -isoprostane), validated markers of metabolic risk factors, fitness and body composition were measured in African American (n = 82) and White American (n = 76) youth (8-17 years old) recruited over a range of BMI percentiles (4th to 99th). RESULTS: F2 -isoprostane concentration was positively correlated with percentage body fat (r = 0.198) and percentage truncal fat (r = 0.173), but was not different between African American and White American males and females (P = 0.208). African American youth had significantly higher mean systolic and diastolic blood pressure (P = 0.023 and P = 0.011, respectively), body weight, BMI percentile and Tanner stage. After adjusting for gender, age, BMI and Tanner stage, African American youth varied from White Americans in the association of F2 -isoprostane with diastolic blood pressure (P = 0.047), but not with systolic blood pressure, triglycerides, VO2PEAK or homeostatic model assessment for insulin resistance (all P > 0.05). CONCLUSIONS: Oxidative stress, as measured by urinary F2 -isoprostane concentrations, was positively associated with percent body fat and truncal fat in youth. Oxidative stress levels were similar among African American and White American youth. Among markers of the metabolic syndrome, a significant difference between African American and White American youth was demonstrated only in the association of oxidative stress with diastolic blood pressure.

Impact of macrophage toll-like receptor 4 deficiency on macrophage infiltration into adipose tissue and the artery wall in mice.

AIMS/HYPOTHESIS: Toll-like receptor 4 (TLR4) is a receptor for saturated fatty acids (SFAs), global deficiency of which has been shown to protect against inflammation, insulin resistance and atherosclerotic lesion formation. Because macrophages express Tlr4 and are important in insulin resistance and atherosclerotic lesion formation due to their infiltration of white adipose tissue (WAT) and the artery wall, respectively, we hypothesised that deficiency of macrophage TLR4 could protect against these disorders. METHODS: Bone marrow transplantation of agouti, LDL-receptor deficient (A(y)/a; Ldlr (-/-)) mice with marrow from either C57BL/6 or Tlr4 (-/-) mice was performed. Recipient mice with Tlr4 (+/+) marrow (MthetaTLR4(+/+)) or with Tlr4 (-/-) marrow (MthetaTLR4(-/-)) were then placed on one of four diets: (1) low fat; (2) high fat; (3) high fat rich in SFAs (HF(SFA)); and (4) HF(SFA) supplemented with fish oil. RESULTS: There were no differences in body composition or plasma lipids between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice on any of the diets. However, we observed a decrease in some macrophage and inflammatory markers in WAT of female low fat-fed MthetaTLR4(-/-) mice compared with MthetaTLR4(+/+) mice. MthetaTLR4(-/-) mice fed low-fat diet also displayed decreased atherosclerotic lesion area. There were no differences in macrophage accrual in WAT or atherosclerosis between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed any of the high-fat diets. Finally, no difference was seen in insulin sensitivity between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed the HF(SFA) diet. CONCLUSIONS/INTERPRETATION: These data suggest that under certain dietary conditions, macrophage expression of Tlr4 can be an important mediator of macrophage accumulation in WAT and the artery wall.

Contrasting net primary productivity and carbon distribution between neighboring stands of Quercus robur and Pinus sylvestris.

Standing biomass, net primary production (NPP) and soil carbon (C) pools were studied in a 67-year-old pedunculate oak (Quercus robur L.) stand and a neighboring 74-year- old Scots pine (Pinus sylvestris L.) stand in the Belgian Campine region. Despite a 14% lower tree density and a lower tree height in the oak stand, standing biomass was slightly higher than in the pine stand (177 and 169 Mg ha(-1) in oaks and pines, respectively), indicating that individual oak trees contained more biomass than pine trees of similar diameter. Moreover, NPP in the oak stand was more than double that in the pine stand (17.7 and 8.1 Mg ha(-1) year(-1), respectively). Several observations indicated that soil organic matter accumulated at higher rates under pines than under oaks. We therefore hypothesized that the pines were exhibiting an age-related decline in productivity due to nutrient limitation. The poor decomposability of pine litter resulted in the observed accumulation of organic matter. The subsequent immobilization of nutrients in the organic matter, combined with the already nutrient-poor soil conditions, resulted in a decrease in total NPP over time, as well as in a substantial shift in the allocation of NPP toward fine roots. In the oak stand, litter is less recalcitrant to decay and soil acidity is less severe; hence, organic matter does not accumulate and nutrients are recycled. This probably explains why NPP was much higher in the oaks than in the pines and why only a small proportion of NPP was allocated to oak fine roots.

The use of genomics and proteomics to understand oocyte and early embryo functions in farm animals.

Oocyte maturation, a simple and visible phenomenon, is about to be transformed into a complex and not so visible molecular cascade leading to the marking of the following generation. The study of oocyte maturation in mammals is progressively changing towards a more molecular approach. This review addresses the main challenges in the study of RNA extraction and quantification in oocytes and embryos as well as the importance of the mRNA maturation. The identification of specific genes in oocytes and embryos is now possible with the use of powerful tools, such as library analysis or subtractions, DNA array, comparative analysis of databanks from other mammals or animals and two-dimensional gel electrophoresis analysis. Finally, RNA interference is a useful tool for studying gene function by knocking out the activity of specific genes and will be used in oocytes and embryos.

The influence of cAMP before or during bovine oocyte maturation on embryonic developmental competence.

This study was designed to evaluate the effects of pretreatment with various forms of cAMP before or during bovine oocyte maturation on the acquisition of embryonic developmental competence. The objective of the 4 experiments was to induce differentiation of the early maturing oocyte in conditions of maintained meiotic arrest or normal maturation. To promote differentiation, different forms of cyclic AMP-dependent protein kinase (PKA) pathways were investigated. The factors studied included follicular fluid, invasive adenylate cyclase (iAC), dibutyryl-cAMP (dbcAMP) and 3-isobutyl-1-methyl-xanthine (IBMX) with or without cycloheximide (CHX). High concentrations of iAC pretreatment were beneficial to the oocyte competence in BSA-iAC maturation while harmful in normal maturation. Also, after 2 to 3 h IBMX-iAC pretreatment, another 6 h of CHX treatment with or without iAC was harmful to the embryonic developmental competence of fertilized oocytes even though it did not have any effect on cleavage rate. Experiment 4 was to assess the role of cAMP in acquisition of oocyte developmental competence before meiotic resumption. Results supported that the intracellular cAMP concentration during the interval between oocyte isolation from the follicle and the beginning of in vitro maturation is critical for requiring optimal developmental competence.

Properties of herbage in relation to equine dysautonomia: biochemical composition and antioxidant and prooxidant actions.

To investigate the etiology of equine dysautonomia (ED), a degenerative polyneuropathy affecting grazing horses, the biochemical composition and antioxidant/prooxidant activities of aqueous extracts of plants collected from ED pastures were determined. Plants collected immediately after an outbreak of ED had reduced antioxidant and weak prooxidant activities when compared with control plants (plants collected from ED pastures out of ED season and control plants from ED pastures that were grown under favorable conditions). ED plants also had significantly increased concentrations of fructose and low molecular weight phenolic compounds, significantly more of one amino acid zone (probably valine), significantly less tartaric acid, and a nonsignificant decrease in ascorbic acid content when compared with control plants from ED pastures that were grown under favorable conditions. These findings suggest that ED plants may be under oxidative stress, possibly due to chilling, drought, or fungal colonization. However, experimental drought and chilling of plants did not reproduce the biochemical alterations identified in ED plants. It is possible that the altered biochemical content of ingested plants may contribute, directly or indirectly, to the development of ED in grazing horses.

The time interval between FSH administration and ovarian aspiration influences the development of cattle oocytes.

Depriving the ovary of exogenous FSH for 1, 2 or 3 d following a bolus injection of FSH was shown to influence the quality of the recovered oocytes. Thus, we compared the developmental competence of oocytes from heifers which had been stimulated for 3 d with FSH (Folltropin-V) and, after an interval of 36, 48 or 60 h, underwent blind transvaginal aspiration. The ovaries of heifers with a palpable or functional corpus luteum were aspirated to remove all large follicles 2 d prior to being injected with either 6 doses of saline (S), 6 doses (20 mg/mL) of FSH (F), or in 6 decreasing doses of FSH (3, 3, 2, 2, 1, 1 mL; Fd). Follicles were counted and classified (medium: 5 to 10 mm, large: >10 mm) with ultrasonography before each aspiration. The oocytes recovered were classified, matured, fertilized, and developed in vitro. On a per animal basis, 1.5, 5.2 and 4.7 large and 1.5, 10.7 and 10.7 medium follicles were counted for S, F and Fd, respectively. A mean of 3.3, 9.1 and 7.7 oocytes was recovered for treatments S, F and Fd, respectively and 58, 94 and 82% were enclosed in a nonexpanded cumulus or a corona layer. Oocyte development rates were based on counts of embryos with 32 or more nuclei at Day 6.5. When oocytes were recovered 36 h after the last injection, an average of 1, 2.7 and 2 embryos per animal was obtained with S, F and Fd, respectively; at 48 h, 0.75, 4.25 and 1 embryo; and at 60 h, 0, 2.5 and 2.7 embryos. Variance analysis was performed, and the protected LSD test indicated that treatment F at 48 h resulted in a significantly higher embryo rate than Fd at 48 h (P<0.05) or S (all times; P<0.05). The reduced effect of the Fd regimen could be due to the decreasing FSH support during follicular growth or to the lower total amount of FSH given. In conclusion, these results indicate an advantage of using moderate (3 d) follicle stimulation followed by a period of FSH starvation to obtain optimal embryo production.

The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation.

The objective of this study was to examine the effects of reactive oxygen species (ROS) on bovine sperm function and on the developmental competence of in vitro-matured bovine oocytes. In a first series of experiments, spermatozoa were exposed to ROS generated through the use of the hypoxanthine-xanthine oxidase system +/- catalase prior to the conduct of in vitro fertilization (IVF). Reactive oxygen species exposure reduced significantly (P < 0.001) the rates of oocyte penetration (control: 56% +/- 4 SEM; ROS: 16 +/- 2-23% +/- 7 SEM), and this effect was reversed by adding catalase (ROS+catalase: 67% +/- 0.3 SEM). During IVF, addition of superoxide dismutase (SOD: 1, 10, or 100 U/ml) had no effect on penetration rates. However, increasing concentrations of catalase (0.1 or 1 mg/ml) reduced these rates significantly (control: 70% +/- 3 SEM; treated: 45% +/- 5 and 1% +/- 1 SEM; P < 0.001). In a second series of experiments, when oocytes were matured in vitro in the presence of exogenous antioxidants (SOD: 10, 100, or 1000 U/ml; beta-mercaptoethanol: 0.01, 0.1, or 0.5 mM; ascorbic acid: 0.05 mg/ml), the developmental competence of the oocytes after IVF was not significantly improved. On the other hand, presumed production of ROS using the hypoxanthine-xanthine system at the beginning of the in vitro maturation period did improve subsequent developmental competence of the oocytes under some conditions and when catalase was present (control: 14% +/- 4 SEM and treated: 23% +/- 9 and 27% +/- 8 SEM; P < 0.05). These observations demonstrate that ROS may be beneficial to gamete function under specific conditions.

In vitro production of bovine embryos: developmental competence is acquired before maturation.

Few studies have examined the importance of the time during which oocytes are left in the ovaries following animal slaughter. The objective of this study was to determine the optimal time for retrieving oocytes after slaughter and to ascertain if superovulating cows in association with this optimal time could increase the developmental competence of bovine oocytes. In Experiment 1, oocytes were left in the postmortem ovaries for 2,3,4,5,6 or 7 h and were then transported to the laboratory at approximately 30 degrees C. Recovered oocytes were processed in vitro using standard techniques. In Experiment 2, cyclic heifers (n = 18) were superovulated between Days 8 and 12 of the estrous cycle with 8 constant doses (4 mg each, twice daily) or 8 decreasing doses (2 injections of 4,3,2 and 1 mg every 12 h) of FSH-P +/- 1 mg prostaglandin 24 or 48 h before slaughter. Oocytes were left in the ovaries for 4 h and were classified according to the state of their cumulus and cytoplasm. The results indicated that oocytes aspirated from ovaries collected 4 h after slaughter produced significantly more > or =64-cell embryos after 7 d of in vitro development than those collected 2, 6 or 7 h postslaughter. Oocytes (87%) from superovulated animals had numerous layers of cumulus cells and originated from medium (2.7 to 8 mm) and large (> or =8 mm) follicles. Significantly more oocytes developed from large follicles than from medium follicles. Although individual culture of the oocytes negatively affected the percentage of embryos produced, group culture of oocytes from animals that were superovulated and left in the postmortem ovaries for 4 h resulted in exceptionally high rates of embryos after 5 d of IVD. On average, 60 to 80% of 16-cell embryos were produced, indicating that under the proper conditions, developmental competence is acquired before in vitro maturation.

Superovulation can reduce the developmental competence of bovine embryos.

This study was done to determine if different superovulatory regimens could have an effect on the percentage of embryos produced using IVM/IVF/IVC. Cyclic heifers (n = 22) were superovulated between Days 8 and 12 of the estrous cycle with 4, 6 or 8 constant doses of FSH-P (4 mg each, twice daily) +/- the addition of 1 mg prostaglandin 24 h before slaughter. Ovaries from these superovulated cows and from untreated cows were collected and the follicles dissected. Oocytes were classified according to the appearance of their cumulus and cytoplasm. Individual culture as well as group culture were performed but an individual culture reduced the percentage of oocytes developing into embryos for both untreated and superovulated animals. The results indicated that despite the superovulation regimen the developmental competence of the oocytes collected was lower (0 to 15% embryos) than that of oocytes from untreated animals (20 to 34% embryos). Small follicles ( < or = 2.7 mm) yielded mostly oocytes with an incomplete or partially expanded cumulus investment that never developed into an embryo. Differences in the morphology of the oocytes from medium (2.7 to 8 mm) and large ( > or = 8 mm) follicles were apparent, but equal developmental rates were obtained between all classes of oocytes (12 and 8% embryos, respectively). Follicular atresia was reduced significantly after superovulation (81% nonatretic follicles in treated vs 42% nonatretic follicles in untreated animals); however oocytes from atretic and slightly atretic follicles developed similarly to those from nonatretic follicles. These results suggest that although superovulation increases follicular size and decreases atresia, these conditions are not sufficient to confer developmental competence on the oocytes.

Selective follicular reduction: what to do with the oocytes?

OBJECTIVE: To investigate the possible benefits of in vitro fertilization (IVF) of oocytes retrieved during selective follicular reduction of supernumerary follicles in non-IVF cycles. METHODS: Selective follicular reduction of supernumerary follicles was used to prevent ovarian hyperstimulation syndrome and multiple pregnancies in gonadotropin-stimulated cycles. We analyzed the data of 13 cycles (13 women) retrospectively. RESULTS: Three pregnancies occurred in these 13 cycles (23%), one after intra-uterine insemination and two after timed coitus. In all cycles, oocytes were retrieved, and in 10 cycles fertilization was achieved (77%); in 6 cycles cryo-preservation was successful (46%) and in 3 cycles embryo transfer (ET) was performed (23%). All embryos were of poor quality and no pregnancies occurred after ET of frozen-thawed embryos. The diagnostic value of fertilization failure seems to be low, since one of the patients who failed to show fertilization became spontaneously pregnant afterward. CONCLUSION: Based on our observations, the beneficial effect of IVF/cryopreservation during selective follicular reduction appears questionable.

The co-culture of cumulus-enclosed bovine oocytes and hemi-sections of follicles: Effects on meiotic resumption.

To prolong the culture of oocytes, it is essential to know how the follicle maintains meiotic arrest. This study was undertaken to evaluate the short-term effects (24 h) of the co-culture of follicular hemi-sections, including theca and granulosa cells, with cumulus-enclosed primary oocytes on meiotic resumption. Bovine oocytes were collected from 1 to 5-mm follicles from ovaries kept at 35 degrees C. Follicular hemi-sections were prepared by careful dissection of another group of follicles of the same size but from ovaries transported on ice. Following 24 h of co-culture, the oocytes were either fixed for determination of nuclear maturation or matured for an additional 24 h to evaluate reversibility of inhibition. The inhibitory action of the hemi-sections on meiotic resumption of oocytes was directly related to the amount of tissue and did not require direct physical contact between the cumulus and the follicular wall. The inhibition was reversible after 24 h of co-culture. Therefore, follicular tissue can be used to maintain meiotic arrest for at least 24 h, thus allowing for the study of changes in developmental competence during late folliculogenesis.

Follicle-oocyte-sperm interactions in vivo and in vitro in pigs.

In vitro culture has provided new information on the mechanisms involved in fertilization where two completely different cells fuse together. At the same time, results obtained in vitro have led to new questions. Does the follicle influence the final maturation process of the oocyte and does the oviduct regulate the normal behaviour of spermatozoa? Recent studies indicate a critical influence of both the follicular compartment and the oviduct on the normal fertilization process. Oocytes matured in vivo are more competent to induce pronuclear formation, but are still susceptible to polyspermy. Oocytes matured in vitro can develop fully after fertilization, but require the presence of follicular factors during culture to enhance their developmental competence. Fresh or frozen spermatozoa can penetrate oocytes in vitro, but their conditioning by differential centrifugation or oviductal cells influences the rate of polyspermy. The understanding of these influences is a prerequisite to enhancing in vitro production of pig embryos.

Modulation of dopamine receptors by cations in 7315a, MtTW15 and estradiol-induced pituitary tumors.

Modulation of dopamine (DA) receptors by cations (Na+, K+, Mg2+, Ca2+) was compared in 7315a, MtTW15, and estradiol valerate-induced (EV-T) pituitary tumors, and intact adenohypophysis. In 7315a tumors, the affinity of [3H]spiperone binding measured at 25 degrees remained unchanged in the presence of each cation individually or all these cations together (IONS) compared to the affinity obtained using a buffer without ions; the density (Bmax) was not affected by monovalent cations or Mg2+ and was decreased by Ca2+ or IONS. When binding experiments were done at 37 degrees, monovalent cations increased affinity whereas divalent cations or IONS did not modify it, and none of these cations affected Bmax values. In MtTW15 tumors, the affinity of [3H]spiperone binding measured at 25 degrees was not changed by Na+ or IONS and was decreased by K+ or divalent cations; the density was decreased by K+ and unchanged by all the other cations. When binding experiments were done at 37 degrees, Na+ increased the affinity, whereas all the other cations did not affect it: the density was unaffected by all the cations studied. In EV-T assayed at 37 degrees, the affinity was increased by monovalent cations or Mg2+ and was unchanged by Ca2+; monovalent cations did not affect the density of [3H]spiperone binding and divalent cations increased it. In binding experiments performed at 25 degrees and 37 degrees, choline chloride did not change the affinity or the density of [3H]spiperone binding to DA receptors in the three pituitary tumors investigated, suggesting that the effect of cations was specific and not due to differences in ionic strength. In the rat normal anterior pituitary, Na+ increased the affinity of [3H]spiperone for the DA receptors, whereas the affinity was unchanged by Ca2+; the density of [3H]spiperone binding was unaffected by these cations. Our results suggest that DA receptors in 7315a and MtTW15 tumors are regulated abnormally by sodium, potassium, magnesium and calcium. In contrast, DA receptors in EV-T are regulated normally by monovalent cations and abnormally by divalent cations as compared to these receptors in intact pituitary tissue.