Maternal high-fat diet changes DNA methylation in the early embryo by disrupting the TCA cycle intermediary alpha ketoglutarate.

In brief: Maternal obesity can impair metabolism in the embryo and the resulting offspring. This study shows that metabolic disruptions through α-ketoglutarate may link altered metabolism with epigenetic changes in embryos. Abstract: Maternal obesity can impair offspring metabolic health; however, the precise mechanism underpinning programming is unknown. Ten-Eleven translocase (TET) enzymes demethylate DNA using the TCA cycle intermediary α-ketoglutarate and may be involved in programming offspring health. Whether TETs are disrupted by maternal obesity is unknown. Five to six week-old C57Bl/6 female mice were fed a control diet (CD; 6% fat, n = 175) or a high-fat diet (HFD; 21% fat, n = 158) for 6 weeks. After superovulation, oocytes were collected for metabolic assessment, or females were mated and zygotes were cultured for embryo development, fetal growth, and assessment of global DNA methylation (5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC)) in the two-cell embryo. Zygotes collected from superovulated CBAF1 females were cultured in media containing α-ketoglutarate (0, 1.4, 3.5, or 14.0 mM) or with 2-hydroxyglutarate (2HG) (0 or 20 mM), a competitive inhibitor of α-ketoglutarate, with methylation and blastocyst differentiation assessed. After HFD, oocytes showed increased pyruvate oxidation and intracellular ROS, with no changes in Tet3 expression, while two-cell embryo global 5hmC DNA methylation was reduced and 5fC increased. Embryos cultured with 1.4 mM α-ketoglutarate had decreased two-cell 5mC, while 14.0 mM α-ketoglutarate increased the 5hmC:5mC ratio. In contrast, supplementation with 20 mM 2HG increased 5mC and decreased 5fC:5mC and 5caC:5mC ratios. α-ketoglutarate up to 3.5 mM did not alter embryo development, while culturing in 14.0 mM α-ketoglutarate blocked development at the two-cell. Culture with 2HG delayed embryo development past the four-cell and decreased blastocyst total cell number. In conclusion, disruptions in metabolic intermediates in the preimplantation embryo may provide a link between maternal obesity and programming offspring for ill health.

The X-linked acrogigantism-associated gene gpr101 is a regulator of early embryonic development and growth in zebrafish.

We recently described X-linked acrogigantism (X-LAG), a condition of early childhood-onset pituitary gigantism associated with microduplications of the GPR101 receptor. The expression of GPR101 in hyperplastic pituitary regions and tumors in X-LAG patients, and GPR101's normally transient pituitary expression during fetal development, suggest a role in the regulation of growth. Nevertheless, little is still known about GPR101's physiological functions, especially during development. By using zebrafish models, we investigated the role of gpr101 during embryonic development and somatic growth. Transient ectopic gpr101 expression perturbed the embryonic body plan but did not affect growth. Loss of gpr101 led to a significant reduction in body size that was even more pronounced in the absence of maternal transcripts, as well as subfertility. These changes were accompanied by gastrulation and hypothalamic defects. In conclusion, both gpr101 loss- and gain-of-function affect, in different ways, fertility, embryonic patterning, growth and brain development.

The effect of methionine and folic acid administered in ovo on the hematological parameters of chickens (Gallus gallus domesticus).

Methionine (Met), an essential amino acid in poultry diets, when overdosed may cause hyperhomocysteinemia, which is mainly a trigger for cardiovascular diseases in humans. Homocysteine is neutralized (remethylated) in the presence of folic acid (FA), which also plays an important role in hematopoiesis and participates in the synthesis of DNA, and its deficiencies may result in the development of neural tube defects. One of the basic tools in studying the impact of both xenobiotics and nutrients on the animal organism is hematological analysis. Thus, the aim of this study was to determine the effect of in ovo supplementation with Met and FA on the hematological parameters of broiler chickens. On the 17th day of incubation, embryonated eggs (Ross 308) were injected with 5 or 25 mg of Met per egg (M5 and M25), 3 and 15 mg of FA per egg (F3 and F15), or a mixture of these 2 compounds (M5/F3 and M25/F15). The broilers were reared in accordance with welfare regulations and fed with commercial diets ad libitum. Blood samples were collected on the first, seventh, and 35th day of rearing (D1, D7, and D35), and complete hematological analysis was performed. The observed changes in red blood cell parameters probably result from physiological changes occurring during bird growth. Mean erythrocyte volume decreased with the age of chickens in the control, M5, and M25 groups, but not in those supplied with FA. Among supplemented groups, the number of white blood cells on D1 was lower only in group M5 than in the sham (C) group. The analysis of leukograms showed no significant differences between the groups. Comparing D1 with D7 in the group injected with a higher dose of Met and FA (MF25/15), a statistically significant increase in the percentage of lymphocytes and a significant decrease in the percentage of heterophils were observed. In addition, in the group injected with a higher FA dose (F15), there was statistically significant reduction in the percentage of eosinophils and a significant increase in the percentage of monocytes at day 7 compared with day 1. It seems that Met supplementation led to temporary immunosuppression in the animals.

New advances and future directions in plant polyspermy.

Plants have evolved a battery of mechanisms that potentially act as polyspermy barriers. Supernumerary sperm fusion to one egg cell has consequently long remained a hypothetical concept. The recent discovery that polyspermy in flowering plants is not lethal but generates viable triploid plants is a game changer affecting the field of developmental biology, evolution, and plant breeding. The establishment of protocols to artificially induce polyspermy together with the development of a high-throughput assay to identify and trace polyspermic events in planta now provide powerful tools to unravel mechanisms of polyspermy regulation. These achievements are likely to open new avenues for animal polyspermy research as well, where forward genetic approaches are hampered by the fatal outcome of supernumerary sperm fusion.

Sericin enhances the developmental competence of heat-stressed bovine embryos.

We investigated the effects of sericin on the developmental competence of bovine embryos exposed to heat stress (HS). Putative zygotes were cultured with sericin and subjected to HS (40.5°C for 6 hr) on Day 2 or 7 followed by continuous culture at 38.5°C until Day 8. Day 2 HS significantly decreased blastocyst development on Day 8 as well as mitochondrial activity, and significantly increased the amount of intracellular reactive oxygen species and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive cells, whereas Day 7 HS only significantly decreased mitochondrial activity and increased the number of TUNEL-positive cells in Day 8 blastocysts. These detrimental effects were neutralized by sericin supplementation. Next, to investigate the potential production of blastocysts with high viability in terms of thermotolerance, embryos were cultured with sericin until Day 7, and then exposed to HS in the sericin-free medium. TUNEL-positive cell numbers were significantly lower in blastocysts produced by sericin culture than in control blastocysts. Transcript abundance for HSPA1A and BAX was significantly decreased but IFNT2 levels were increased in blastocysts produced by sericin culture. In conclusion, these findings demonstrate the anti-oxidative and anti-apoptotic activities of sericin, and the potential use of sericin to produce embryos with high viability in vitro.

Maternal allocation of carotenoids to eggs in an Anolis lizard.

The maternal allocation of carotenoids to eggs has been widely documented and manipulated. However, it is often assumed that the sole adaptive value of this allocation is to increase offspring fitness. Because carotenoids can be pro-oxidants or antioxidants depending on their concentrations and their chemical environment (i.e. presence of other antioxidants), dams may need to dispose of excess carotenoids upon depletion of other antioxidants to prevent oxidative damage. Additionally, the amount of carotenoids deposited in eggs may be dependent on male traits such as quality and coloration. We evaluated these two non-mutually exclusive hypotheses for carotenoid allocation to eggs and assessed paternal effects by supplementing male and female brown anole lizards, Anolis sagrei, with dietary carotenoids or with a combination of carotenoids and vitamin C. We found significant differences in the antioxidant capacities of fertilized and unfertilized eggs produced by female lizards, but the treatment did not affect the antioxidant capacity or carotenoid content of eggs. However, the carotenoid concentration of unfertilized eggs from carotenoid-supplemented females was significantly higher than eggs from the control group. Male coloration and body size did not affect the antioxidant capacity or carotenoid content of the eggs. Carotenoids may be allocated to unfertilized eggs to offset oxidative damage to the dam, with a neutral effect on offspring, rather than to solely provide antioxidant benefits to offspring as has been widely assumed.

Pollen germination and in vivo fertilization in response to high-temperature during flowering in hybrid and inbred rice.

High-temperature during flowering in rice causes spikelet sterility and is a major threat to rice productivity in tropical and subtropical regions, where hybrid rice development is increasingly contributing to sustain food security. However, the sensitivity of hybrids to increasing temperature and physiological responses in terms of dynamic fertilization processes is unknown. To address these questions, several promising hybrids and inbreds were exposed to control temperature and high day-time temperature (HDT) in Experiment 1, and hybrids having contrasting heat tolerance were selected for Experiment 2 for further physiological investigation under HDT and high-night-time-temperature treatments. The day-time temperature played a dominant role in determining spikelet fertility compared with the night-time temperature. HDT significantly induced spikelet sterility in tested hybrids, and hybrids had higher heat susceptibility than the high-yielding inbred varieties. Poor pollen germination was strongly associated with sterility under high-temperature. Our novel observations capturing the series of dynamic fertilization processes demonstrated that pollen tubes not reaching the viable embryo sac was the major cause for spikelet sterility under heat exposure. Our findings highlight the urgent need to improve heat tolerance in hybrids and incorporating early-morning flowering as a promising trait for mitigating HDT stress impact at flowering.

Glutathione peroxidase 4 inhibits Wnt/ß-catenin signaling and regulates dorsal organizer formation in zebrafish embryos.

The Wnt/ß-catenin signaling pathway plays pivotal roles in axis formation during embryogenesis and in adult tissue homeostasis. Glutathione peroxidase 4 (GPX4) is a selenoenzyme and participates in the reduction of peroxides. Its synthesis depends on the availability of the element selenium. However, the roles of GPX4 in vertebrate embryonic development and underlying mechanisms are largely unknown. Here, we show that maternal loss of zebrafish gpx4b promotes embryonic dorsal organizer formation, whereas overexpression of gpx4b inhibits the development of the dorsal organizer. Depletion of human GPX4 and zebrafish gpx4b (GPX4/gpx4b) increases, while GPX4/gpx4b overexpression decreases, Wnt/ß-catenin signaling in vivo and in vitro Functional and epistatic studies showed that GPX4 functions at the Tcf/Lef level, independently of selenocysteine activation. Mechanistically, GPX4 interacts with Tcf/Lefs and inhibits Wnt activity by preventing the binding of Tcf/Lefs to the promoters of Wnt target genes, resulting in inhibitory action in the presence of Wnt/ß-catenin signaling. Our findings unravel GPX4 as a suppressor of Wnt/ß-catenin signals, suggesting a possible relationship between the Wnt/ß-catenin pathway and selenium via the association of Tcf/Lef family proteins with GPX4.

Starch Turnover and Metabolism during Flower and Early Embryo Development.

The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed.

Inhibitory effect of cadmium on estrogen signaling in zebrafish brain and protection by zinc.

The present study was conducted to assess the effects of Cd exposure on estrogen signaling in the zebrafish brain, as well as the potential protective role of Zn against Cd-induced toxicity. For this purpose, the effects on transcriptional activation of the estrogen receptors (ERs), aromatase B (Aro-B) protein expression and molecular expression of related genes were examined in vivo using wild-type and transgenic zebrafish embryos. For in vitro studies, an ER-negative glial cell line (U251MG) transfected with different zebrafish ER subtypes (ERα, ERß1 and ERß2) was also used. Embryos were exposed either to estradiol (E2 ), Cd, E2 +Cd or E2 +Cd+Zn for 72 h and cells were exposed to the same treatments for 30 h. Our results show that E2 treatment promoted the transcriptional activation of ERs and increased Aro-B expression, at both the protein and mRNA levels. Although exposure to Cd, does not affect the studied parameters when administered alone, it significantly abolished the E2 -stimulated transcriptional response of the reporter gene for the three ER subtypes in U251-MG cells, and clearly inhibited the E2 induction of Aro-B in radial glial cells of zebrafish embryos. These inhibitory effects were accompanied by a significant downregulation of the expression of esr1, esr2a, esr2b and cyp19a1b genes compared to the E2 -treated group used as a positive control. Zn administration during simultaneous exposure to E2 and Cd strongly stimulated zebrafish ERs transactivation and increased Aro-B protein expression, whereas mRNA levels of the three ERs as well as the cyp19a1b remained unchanged in comparison with Cd-treated embryos. In conclusion, our results clearly demonstrate that Cd acts as a potent anti-estrogen in vivo and in vitro, and that Cd-induced E2 antagonism can be reversed, at the protein level, by Zn supplement. Copyright © 2016 John Wiley & Sons, Ltd.

Genotype Reconstruction of Paternity in European Lobsters (Homarus gammarus).

Decapod crustaceans exhibit considerable variation in fertilisation strategies, ranging from pervasive single paternity to the near-ubiquitous presence of multiple paternity, and such knowledge of mating systems and behaviour are required for the informed management of commercially-exploited marine fisheries. We used genetic markers to assess the paternity of individual broods in the European lobster, Homarus gammarus, a species for which paternity structure is unknown. Using 13 multiplexed microsatellite loci, three of which are newly described in this study, we genotyped 10 eggs from each of 34 females collected from an Atlantic peninsula in the south-western United Kingdom. Single reconstructed paternal genotypes explained all observed progeny genotypes in each of the 34 egg clutches, and each clutch was fertilised by a different male. Simulations indicated that the probability of detecting multiple paternity was in excess of 95% if secondary sires account for at least a quarter of the brood, and in excess of 99% where additional sire success was approximately equal. Our results show that multiple paternal fertilisations are either absent, unusual, or highly skewed in favour of a single male among H. gammarus in this area. Potential mechanisms upholding single paternal fertilisation are discussed, along with the prospective utility of parentage assignments in evaluations of hatchery stocking and other fishery conservation approaches in light of this finding.

Mitochondrial replacement therapy in reproductive medicine.

Mitochondrial dysfunction is implicated in disease and age-related infertility. Mitochondrial replacement therapies (MRT) in oocytes or zygotes, such as pronuclear (PNT), spindle (ST), or polar body (PBT) transfer, could prevent second-generation transmission of mitochondrial DNA (mtDNA) defects. PNT, associated with high levels of mtDNA carryover in mice but low levels in human embryos, carries ethical issues secondary to donor embryo destruction. ST, developed in primates, supports normal development to adults and low mtDNA carryover. PBT in mice, coupled with PN or ST, may increase the yield of reconstructed embryos with low mtDNA carryover. MRT also offers replacement of the deficient cytoplasm in oocytes from older patients, with the expectation of high pregnancy rates following in vitro fertilization.

Systematic analysis of the factors that adversely affect the rate of cell accumulation in mouse embryos during their culture in vitro.

BACKGROUND: Retarded embryo growth is a pervasive effect of culture in vitro. METHODS: A systematic analysis of the interactions between media design, embryo culture density, oxygen tension, amino acids, trophic ligands and the genetic background of the mouse on embryo growth rates in vitro was performed. RESULTS: Growth retardation of mouse zygotes was greater in 20% O2 than 5%, a sequential media design was superior to static simple media designs, but the supplementation of simple media with mixed amino acids mitigated this difference. There was a beneficial effect of communal culture in small volumes, and supplementation with a trophic ligand (Paf) further enhanced growth rates. For hybrid strain zygotes (B6CBF1) communal culture in KSOM media supplemented with amino acids, albumin and Paf under 5% O2 resulted in complete rescue of their rate of accumulation of cells and blastocyst formation. Inbred strain (C57BL6/J) zygotes, however, still showed some retardation of development under these conditions. The additional supplementation of media with another trophic ligand (IGF1) showed a further additive beneficial effect on development of inbred strain embryos but they still showed a growth deficit of ~ 23% cell number. The results show that optimising the interactions between a range of culture conditions and media design can rescue hybrid strain embryos from a retarded rate of cell proliferation caused by culture in vitro, but this was incomplete for the B6 strain. CONCLUSIONS: The results indicate that the growth requirement of embryos in vitro varies depending upon their genetic background and provide models for the further genetic analysis of embryo growth.

Dimethylsulfoxide and conjugated linoleic acids affect bovine embryo development in vitro.

Conjugated linoleic acids (CLA) are employed to overcome the bovine periparturitional negative energy balance. Especially of interest are trans10,cis12 -linoleic acid (t10c12-CLA) and cis9,trans11-linoleic acid (c9t11-CLA). Their impact on embryonic development, though, is not clear. Here, effects of both above-mentioned CLA on bovine in vitro-produced embryos were assessed. Zygotes (n=2098) were allocated to one of seven groups: cultured with 50 or 100µM of either c9t11-CLA or t10c12-CLA, with 14 or 28mM DMSO or without supplement (control). Messenger RNA analysis of target gene transcripts (IGF1R, IGFBP2, IGFBP4, CPT2, ACAA1, ACAA2, FASN, SCD) via RT-qPCR was performed in single blastocysts. Cleavage rates did not differ, whereas development rates were decreased in both t10c12-supplemented groups in comparison to the unsupplemented group (31.7% ±2.2 control vs 20.2% ±2.0 50µM t10c12 vs 21.0% ±2.8 100µM t10c12). Compared with the unsupplemented group, SCD was expressed at a lower level in embryos cultured with 50µM c9t11-CLA. The relative amount of several transcripts was increased in embryos cultured with 14mM DMSO in comparison to those that developed in the presence of 50µM t10c12-CLA (IGFBP2, ACAA1, CPT2, FASN, SCD) or 50µM c9t11-CLA (IGF1R, IGFBP2, ACAA1, CPT2, FASN, SCD). The molecular analyses show that CLA influence embryonic fat metabolism.

Among-species differences in pollen quality and quantity limitation: implications for endemics in biodiverse hotspots.

BACKGROUND AND AIMS: Insufficient pollination is a function of quantity and quality of pollen receipt, and the relative contribution of each to pollen limitation may vary with intrinsic plant traits and extrinsic ecological properties. Community-level studies are essential to evaluate variation across species in quality limitation under common ecological conditions. This study examined whether endemic species are more limited by pollen quantity or quality than non-endemic co-flowering species in three endemic-rich plant communities located in biodiversity hotspots of different continents (Andalusia, California and Yucatan). METHODS: Natural variations in pollen receipt and pollen tube formation were analysed for 20 insect-pollinated plants. Endemic and non-endemic species that co-flowered were paired in order to estimate and compare the quantity and quality components of pre-zygotic pollination success, obtained through piecewise regression analysis of the relationship between pollen grains and pollen tubes of naturally pollinated wilted flowers. KEY RESULTS: Pollen tubes did not frequently exceed the number of ovules per flower. Only the combination of abundant and good quality pollen and a low number of ovules per flower conferred relief from pre-zygotic pollen limitation in the three stochastic pollination environments studied. Quality of pollen receipt was found to be as variable as quantity among study species. The relative pollination success of endemic and non-endemic species, and its quantity and quality components, was community dependent. CONCLUSIONS: Assessing both quality and quantity of pollen receipt is key to determining the ovule fertilization potential of both endemic and widespread plants in biodiverse hotspot regions. Large natural variation among flowers of the same species in the two components and pollen tube formation deserves further analysis in order to estimate the environmental, phenotypic and intraindividual sources of variation that may affect how plants evolve to overcome this limitation in different communities worldwide.

Screening for microbial metabolites affecting phenotype of Caenorhabditis elegans.

Microbial samples, including our library of known microbial compounds (ca. 300) and microbial culture broths (ca. 9000), were screened for small molecules affecting the phenotype of Caenorhabditis elegans. As a result, seven known compounds were found to induce phenotypic abnormality of C. elegans. Staurosporine exhibited morphological defects in the vulva and tail of C. elegans, avermectin B1a exhibited hatching inhibition of starting eggs on day 1 at 25-100 µM and growth inhibition at 0.01-12.5 µM, siccanin and antimycin A inhibited the growth of C. elegans, and fluorouracil inhibited hatching of eggs newly spawned by adult C. elegans. Toromycin induced morphological defects in the intestine. 5-(4-Methoxyphenyl)-oxazole, isolated as a fungal metabolite for the first time, inhibited the hatching of eggs newly spawned by adult C. elegans.

Beta-oxidation is essential for mouse oocyte developmental competence and early embryo development.

Oocyte and embryo metabolism are closely linked with their subsequent developmental capacity. Lipids are a potent source of cellular energy, yet little is known about lipid metabolism during oocyte maturation and early embryo development. Generation of ATP from lipids occurs within mitochondria via beta-oxidation of fatty acids, with the rate-limiting step catalyzed by carnitine palmitoyl transferase I (CPT1B), a process also requiring carnitine. We sought to investigate the regulation and role of beta-oxidation during oocyte maturation and preimplantation development. Expression of Cpt1b mRNA, assessed by real-time RT-PCR in murine cumulus-oocyte complexes (COCs), increased following hormonal induction of oocyte maturation and ovulation in vivo with human chorionic gonadotropin (5 IU) and in embryos reaching the blastocyst stage. Beta-oxidation, measured by the production of (3)H(2)O from [(3)H]palmitic acid, was significantly increased over that in immature COCs following induction of maturation in vitro with epidermal growth factor (3 ng/ml) and follicle-stimulating hormone (50 mIU/ml). The importance of lipid metabolism for oocyte developmental competence and early embryo development was demonstrated by assessing the rate of embryo development following inhibition or upregulation of beta-oxidation with etomoxir (an inhibitor of CPT1B) or L-carnitine, respectively. Inhibition of beta-oxidation during oocyte maturation or zygote cleavage impaired subsequent blastocyst development. In contrast, L-carnitine supplementation during oocyte maturation significantly increased beta-oxidation, improved developmental competence, and in the absence of a carbohydrate energy supply, significantly increased 2-cell cleavage. Thus, carnitine is an important cofactor for developing oocytes, and fatty acids are an important energy source for oocyte and embryo development.

Reproductive success, early life stage development, and survival of westslope cutthroat trout (Oncorhynchus clarki lewisi) exposed to elevated selenium in an area of active coal mining.

The effects of accumulated Se on the reproductive success and larval development of cutthroat trout (Oncorhynchus clarki lewis,) collected from a site of active coal mining in British Columbia were assessed. Eggs from 12 fish from an exposed site (Clode Pond) and 16 from a reference site (O'Rourke Lake) were field-collected and reared in the laboratory. Egg Se concentrations ranged from 12.3 to 16.7 and 11.8 to 140.0 microg/g dry weight (dw) from fish collected at the reference and exposed sites, respectively. Other studies, including those with this species, have not shown Se to affect egg viability; however, in the present study, eggs with Se concentrations > 86.3 microg/g dw were not successfully fertilized or were nonviable at fertilization, while eggs with concentrations > 46.8 and < 75.4 microg/g dw were fertilized (96% reached the eyed stage) but did not produce viable fry. A significant positive relationship between egg Se concentration and alevin mortality was observed. Deformities were analyzed in surviving fry which developed from eggs with Se concentrations between 11.8 and 20.6 microg/g dw. No relationship between Se concentration in eggs and deformities or edema was found in this range, suggesting that the no-effect threshold for deformity is > 20.6 microg/g dw. The present data, in conjunction with the data from several other studies in temperate fish, suggest that current Se thresholds are conservative for cold-water fish.

Antioxidants in eggs of great tits Parus major from Chernobyl and hatching success.

Antioxidants are powerful protectors against the damaging effects of free radicals that constitute the inevitable by-products of aerobic metabolism. Growing embryos are particularly susceptible to the damaging effects of free radicals produced during rapid growth, and mothers of many species provide protection against such damage by allocating antioxidants to their eggs. Birds living in radioactively contaminated areas use dietary antioxidants to cope with the damaging effects of radiation, but females also allocate dietary antioxidants to eggs, potentially enforcing a physiological trade-off between self-maintenance and reproductive investment. Here we tested whether female great tits Parus major breeding in radioactively contaminated study areas near Chernobyl allocated less dietary antioxidants to eggs, and whether such reduced allocation of dietary antioxidants to eggs had fitness consequences. Concentrations of total yolk carotenoids and vitamins A and E were depressed near Chernobyl compared to concentrations in a less contaminated Ukrainian study area and a French control study area, and all antioxidants showed dose-dependent relationships with all three dietary antioxidants decreasing with increasing level of radiation at nest boxes. These effects held even when controlling statistically for potentially confounding habitat variables and covariation among antioxidants. Laying date was advanced and clutch size increased at nest boxes with high dose rates. Hatching success increased with increasing concentration of vitamin E, implying that hatching success decreased at boxes with high levels of radiation, eventually eliminating and even reversing the higher potential reproductive output associated with early reproduction and large clutch size. These findings are consistent with the hypothesis that radioactive contamination reduced levels of dietary antioxidants in yolks, with negative consequences for hatching success and reproductive success.