Jim Craigon

DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status

A complex combination of adult health-related disorders can originate from developmental events that occur in utero. The periconceptional period may also be programmable. We report on the effects of restricting the supply of specific B vitamins (i.e., B12 and folate) and methionine, within normal physiological ranges, from the periconceptional diet of mature female sheep. We hypothesized this would lead to epigenetic modifications to DNA methylation in the preovulatory oocyte and/or preimplantation embryo, with long-term health implications for offspring. DNA methylation is a key epigenetic contributor to maintenance of gene silencing that relies on a dietary supply of methyl groups. We observed no effects on pregnancy establishment or birth weight, but this modest early dietary intervention led to adult offspring that were both heavier and fatter, elicited altered immune responses to antigenic challenge, were insulin-resistant, and had elevated blood pressure–effects that were most obvious in males. The altered methylation status of 4% of 1,400 CpG islands examined by restriction landmark genome scanning in the fetal liver revealed compelling evidence of a widespread epigenetic mechanism associated with this nutritionally programmed effect. Intriguingly, more than half of the affected loci were specific to males. The data provide the first evidence that clinically relevant reductions in specific dietary inputs to the methionine/folate cycles during the periconceptional period can lead to widespread epigenetic alterations to DNA methylation in offspring, and modify adult health-related phenotypes.

Amino acid and fatty acid composition of follicular fluid as predictors of in-vitro embryo development

The value of using the amino acid and fatty acid composition of follicular fluid as predictors of embryo development was assessed in a bovine model of in-vitro maturation (IVM), IVF and blastocyst culture (IVC). A total of 445 cumulus-oocyte complexes (COC) aspirated from visually healthy follicles underwent IVM and IVF singly (n = 138) or in groups (n = 307). Of these COC, 349 cleaved (78%) following IVF and 112 went on to form blastocysts (32% of cleaved) following IVC. Culture method (singly or in groups) had no effect on development. In contrast to fatty acids, which had no predictive value, the amino acid composition of follicular fluid was associated with morphological assessments of COC quality and with post-fertilization development to the blastocyst stage. Principal component analysis identified two amino acids (i.e. alanine and glycine) that had the highest value for predicting early post-fertilization development. The predictive value of these two amino acids, in terms of the percentage of oocytes that cleaved following IVF, was greatest for COC with the poorest morphological grades but, with respect to blastocyst yields, was independent of morphological grade, and so may serve as a useful additional non-invasive measure of COC quality.

Fetal exposure to a maternal low-protein diet during mid-gestation results in muscle-specific effects on fibre type composition in young rats

This study assessed the impact of reduced dietary protein during specific periods of fetal life upon muscle fibre development in young rats. Pregnant rats were fed a control or low-protein (LP) diet at early (days 0-7 gestation, LPEarly), mid (days 8-14, LPMid), late (days 15-22, LPLate) or throughout gestation (days 0-22, LPAll). The muscle fibre number and composition in soleus and gastrocnemius muscles of the offspring were studied at 4 weeks of age. In the soleus muscle, both the total number and density of fast fibres were reduced in LPMid females (P = 0.004 for both, Diet x Sex x Fibre type interactions), while both the total number and density of glycolytic (non-oxidative) fibres were reduced in LPEarly, LPMid and LPLate (but not LPAll) offspring compared with controls (P < 0.001 for both, Diet x Fibre type interaction). In the gastrocnemius muscle, only the density of oxidative fibres was reduced in LPMid compared with control offspring (P = 0.019, Diet x Fibre type interaction), with the density of slow fibres being increased in LPAll males compared with control (P = 0.024, Diet x Sex x Fibre type interaction). There were little or no effects of maternal diet on fibre type diameters in the two muscles. In conclusion, a maternal low-protein diet mainly during mid-pregnancy reduced muscle fibre number and density in 4-week-old rats, but there were muscle-specific differences in the fibre types affected.

Uptake of Metals by Plants Sharing a Rhizosphere with the Hyperaccumulator Thlaspi caerulescens

ABSTRACT The hyperaccumulator Thlaspi caerulescens was grown with Hordeum vulgare and Lepidium heterophyllum in a split pot experiment to examine the effect of rhizosphere interactions on metal uptake. The objective was to assess the viability of such intercropping as either (1) a system of ‘phytoprotection’ for nonaccumulating plants or (2) a means of enhancing phytoextraction with large-biomass crops through increased metal mobilization within the shared rhizosphere. The plants were separated by (1) an impermeable barrier, (2) a permeable root barrier, or (3) no physical barrier to allow different degrees of root interaction. Studies of rhizosphere effects using split pot experiments are subject to considerable uncertainty by the need to relate test results to appropriate control plants. This was resolved by comparing plant metal concentrations to ‘equivalent’ control plants, with the same yield, based on the observed variation in metal concentration with yield under similar growing conditions. Cadmium concentration in H. vulgare was increased by a factor of 2.4 when it was grown alongside T. caerulescens without a barrier. In contrast, the uptake of zinc by H. vulgare was significantly decreased, probably through metal depletion within the zone of the Zn-hyperaccumulators rhizosphere. T. caerulescens also apparently increased the concentration of Cd in H. vulgare by a factor of 1.4 when the roots of the two plants were separated by a permeable barrier that allowed movement of soil solution but prevented physical mixing of roots. The concentrations of all the metals studied (Cd, Zn, Cu, Pb, Ni) were greater in T. caerulescens when the hyperaccumulator was grown alongside either L. heterophyllum or H. vulgare without a root barrier — probably through successful exploitation of a greater volume of soil. However, this effect was not seen in the presence of a partial barrier, except in the case of Cu when T. caerulescens was grown alongside H. vulgare. These results suggest that T. caerulescens may alter conditions in shared rhizospheres and thereby affect the availability of selected metals to neighboring plants. Thus, it is possible that under-sowing some plants with small hyperaccumulators may potentially offer an alternative form of management for marginally contaminated soils. There was limited evidence of an intercropped hyperaccumulator mobilizing selected metals and restricting the availability of others. However, changes in uptake of selected metals by the larger plant may be quite small compared with the requirements of crop protection or the short-term requirements of many land remediation programs.

Effects of elevated carbon dioxide and ozone on the growth and yield of potatoes (Solanum tuberosum) grown in open-top chambers.

Potato (Solanum tuberosum cv. Bintje) was grown in open-top chambers under three carbon dioxide (ambient and seasonal mean concentrations of 550 and 680 mumol mol-1 CO2) and two ozone concentrations (ambient and an 8 h day-1 seasonal mean of 50 nmol mol-1 O3) between emergence and final harvest. Periodic non-destructive measurements were made and destructive harvests were carried out at three key developmental stages (24, 49 and 101 days after emergence) to establish effects on growth and tuber yield. Season-long exposure to elevated O3 reduced above-ground dry weight at final harvest by 8.4% (P < 0.05), but did not affect tuber yields. There was no significant interaction between CO2 and O3 for any of the growth and yield variables examined. Non-destructive analyses revealed no significant effect of elevated CO2 on plant height, leaf number or green leaf area ratio. However, destructive harvests at tuber initiation and 500 degrees Cd after emergence showed that above-ground dry weight (8 and 7% respectively) and tuber yield (88 and 44%) were significantly increased (P < 0.05) in the 550 mumol mol-1 CO2 treatment. Responses to 550 and 680 mumol mol-1 CO2 were not significantly different for most parameters examined, suggesting the existence of an upper limit to the beneficial influence of CO2 enrichment. Significant effects on above-ground dry weight and tuber yield were no longer apparent at final harvest, although tuber numbers were increased (P < 0.05) under elevated CO2, particularly in the smaller size categories. The results show that the O3 treatment imposed was insufficient to reduce tuber yields and that, although elevated CO2 enhanced crop growth during the early stages of the season, this beneficial effect was not sustained to maturity.

To what extent can zero tillage lead to a reduction in greenhouse gas emissions from temperate soils

Soil tillage practices have a profound influence on the physical properties of soil and the greenhouse gas (GHG) balance. However there have been very few integrated studies on the emission of carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) and soil biophysical and chemical characteristics under different soil management systems. We recorded a significantly higher net global warming potential under conventional tillage systems (26–31% higher than zero tillage systems). Crucially the 3-D soil pore network, imaged using X-ray Computed Tomography, modified by tillage played a significant role in the flux of CO2 and CH4. In contrast, N2O flux was determined mainly by microbial biomass carbon and soil moisture content. Our work indicates that zero tillage could play a significant role in minimising emissions of GHGs from soils and contribute to efforts to mitigate against climate change.

Dynamic Changes in Meiotic Progression and Improvement of Developmental Competence of Pig Oocytes in Vitro by Follicle-Stimulating Hormone and Cycloheximide

Abstract The effects of FSH, LH, and epidermal growth factor (EGF) on the dynamics of nuclear maturation and subsequent embryo development were examined in pig oocytes cultured either conventionally or after preincubation with cycloheximide (CHX). In conventional culture, FSH or EGF significantly increased the rate of attainment of metaphase II (MII) for both gilt (50.0% ± 4.2% and 54.8% ± 4.3%, respectively; control, 5.8% ± 1.8%; P < 0.001) and sow (87.6% ± 3.4% and 78.8% ± 3.9%, respectively; control, 7.8% ± 2.5%; P < 0.001) oocytes. Gilt oocytes treated with both FSH and EGF showed an additive response (93.7% ± 2.1%). Treatment with LH had no effect. Preincubation with CHX caused the majority (84–100%) of both gilt and sow oocytes to undergo germinal vesicle breakdown. Compared to those treated with LH and/or EGF (both >80%), fewer FSH-treated oocytes reached metaphase I (43.8% ± 5.3%, P < 0.001) by 14 h and MII (48.4% ± 5.9%, P < 0.001) by 24 h, although the majority (71%) did mature to MII by 36 h after removal of CHX. After in vitro fertilization, higher proportions of both CHX-pretreated and untreated, FSH-exposed oocytes cleaved (71.3% ± 2.9% and 75.3% ± 3.1%, respectively) compared with those not treated with FSH (37.7% ± 3.0% and 43.0% ± 2.9%, respectively; P < 0.001). Pretreatment with CHX significantly increased blastocyst yield for both FSH-treated (32.8% ± 2.0% and 10.3% ± 1.5%, respectively; P < 0.001) and untreated (16.7% ± 1.5% and 9.4% ± 1.2%, respectively; P < 0.001) oocytes. Polyspermy rates were unaffected. In conclusion, pig oocytes meiotically arrested by CHX before maturation retain and improve their developmental competence. FSH stimulates nuclear maturation but slows meiotic progression.

Photosynthetic responses to elevated CO2and O3 in field-grown potato(Solanum tuberosum)

Summary Potato plants ( Solanum tuberosum L. cv. Bintje) were grown to maturity in open-top chambers under three carbon dioxide (CO 2 ; ambient and 24 h d −1 seasonal mean concentrations of 550 and 680 μmol mol −1 ) and two ozone levels (O 3 ; ambient and an 8 h d −1 seasonal mean of 50 nmol mol −1 ). Chlorophyll content, photosynthetic characteristics, and stomatal responses were determined to test the hypothesis that elevated atmospheric CO 2 may alleviate the damaging influence of O 3 by reducing uptake by the leaves. Elevated O 3 had no detectable effect on photosynthetic characteristics, leaf conductance, or chlorophyll content, but did reduce SPAD values for leaf 15, the youngest leaf examined. Elevated CO 2 also reduced SPAD values for leaf 15, but not for older leaves; destructive analysis confirmed that chlorophyll content was decreased. Leaf conductance was generally reduced by elevated CO 2 , and declined with time in the youngest leaves examined, as did assimilation rate ( A ). A generally increased under elevated CO 2 , particularly in the older leaves during the latter stages of the season, thereby increasing instantaneous transpiration efficiency. Exposure to elevated CO 2 and/or O 3 had no detectable effect on dark-adapted fluorescence, although the values decreased with time. Analysis of the relationships between assimilation rate and intercellular CO 2 concentration and photosynthetically active photon flux density showed there was initially little treatment effect on CO 2 -saturated assimilation rates for leaf 15. However, the values for plants grown under 550 μmol mol −1 CO 2 were subsequently greater than in the ambient and 680 μmol mol −1 treatments, although the beneficial influence of the former treatment declined sharply towards the end of the season. Light-saturated assimilation was consistently greater under elevated CO 2 , but decreased with time in all treatments. The values decreased sharply when leaves grown under elevated CO 2 were measured under ambient CO 2 , but increased when leaves grown under ambient CO 2 were examined under elevated CO 2 . The results obtained indicate that, although elevated CO 2 initially increased assimilation and growth, these beneficial effects were not necessarily sustained to maturity as a result of photosynthetic acclimation and the induction of earlier senescence.

Excess maternal salt intake produces sex-specific hypertension in offspring: putative roles for kidney and gastrointestinal sodium handling.

Hypertension is common and contributes, via cardiovascular disease, towards a large proportion of adult deaths in the Western World. High salt intake leads to high blood pressure, even when occurring prior to birth – a mechanism purported to reside in altered kidney development and later function. Using a combination of in vitro and in vivo approaches we tested whether increased maternal salt intake influences fetal kidney development to render the adult individual more susceptible to salt retention and hypertension. We found that salt-loaded pregnant rat dams were hypernatraemic at day 20 gestation (147±5 vs. 128±5 mmoles/L). Increased extracellular salt impeded murine kidney development in vitro, but had little effect in vivo. Kidneys of the adult offspring had few structural or functional abnormalities, but male and female offspring were hypernatraemic (166±4 vs. 149±2 mmoles/L), with a marked increase in plasma corticosterone (e.g. male offspring; 11.9 [9.3–14.8] vs. 2.8 [2.0–8.3] nmol/L median [IQR]). Furthermore, adult male, but not female, offspring had higher mean arterial blood pressure (effect size, +16 [9–21] mm Hg; mean [95% C.I.]. With no clear indication that the kidneys of salt-exposed offspring retained more sodium per se, we conducted a preliminary investigation of their gastrointestinal electrolyte handling and found increased expression of proximal colon solute carrier family 9 (sodium/hydrogen exchanger), member 3 (SLC9A3) together with altered faecal characteristics and electrolyte handling, relative to control offspring. On the basis of these data we suggest that excess salt exposure, via maternal diet, at a vulnerable period of brain and gut development in the rat neonate lays the foundation for sustained increases in blood pressure later in life. Hence, our evidence further supports the argument that excess dietary salt should be avoided per se, particularly in the range of foods consumed by physiologically immature young.

Adult-Onset Obesity Reveals Prenatal Programming of Glucose-Insulin Sensitivity in Male Sheep Nutrient Restricted during Late Gestation

Background Obesity invokes a range of metabolic disturbances, but the transition from a poor to excessive nutritional environment may exacerbate adult metabolic dysfunction. The current study investigated global maternal nutrient restriction during early or late gestation on glucose tolerance and insulin sensitivity in the adult offspring when lean and obese. Methods/Principal Findings Pregnant sheep received adequate (1.0M; CE, n = 6) or energy restricted (0.7M) diet during early (1–65 days; LEE, n = 6) or late (65–128 days; LEL, n = 7) gestation (term ∼147 days). Subsequent offspring remained on pasture until 1.5 years when all received glucose and insulin tolerance tests (GTT & ITT) and body composition determination by dual energy x-ray absorptiometry (DXA). All animals were then exposed to an obesogenic environment for 6–7 months and all protocols repeated. Prenatal dietary treatment had no effect on birth weight or on metabolic endpoints when animals were ‘lean’ (1.5 years). Obesity revealed generalised metabolic ‘inflexibility’ and insulin resistance; characterised by blunted excursions of plasma NEFA and increased insulinAUC (from 133 to 341 [s.e.d. 26] ng.ml−1.120 mins) during a GTT, respectively. For LEL vs. CE, the peak in plasma insulin when obese was greater (7.8 vs. 4.7 [s.e.d. 1.1] ng.ml−1) and was exacerbated by offspring sex (i.e. 9.8 vs. 4.4 [s.e.d. 1.16] ng.ml−1; LEL male vs. CE male, respectively). Acquisition of obesity also significantly influenced the plasma lipid and protein profile to suggest, overall, greater net lipogenesis and reduced protein metabolism. Conclusions This study indicates generalised metabolic dysfunction with adult-onset obesity which also exacerbates and ‘reveals’ programming of glucose-insulin sensitivity in male offspring prenatally exposed to maternal undernutrition during late gestation. Taken together, the data suggest that metabolic function appears little compromised in young prenatally ‘programmed’ animals so long as weight is adequately controlled. Nutritional excess in adulthood exacerbates any programmed phenotype, indicating greater vigilance over weight control is required for those individuals exposed to nutritional thrift during gestation.