Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.     

Changes in mitochondrial DNA levels during early embryogenesis in Torenia fournieri and Arabidopsis thaliana

Changes in the amount of mitochondrial DNA (mtDNA) have never been investigated in plant zygotes or early plant embryos due to the difficulty in isolating these cells, although such changes have been investigated in mammalian embryos. Using the single‐cell quantitative real‐time polymerase chain reaction (PCR) and laser confocal microscopy, we surveyed the changes in mtDNA levels during early embryogenesis in Torenia fournieri and Arabidopsis thaliana. In contrast with the amount of mtDNA in early mammalian embryos, which does not change, we found that mtDNA doubling occurred during zygotic development in T. fournieri and during two‐cell proembryo development in A. thaliana. These findings reveal that mtDNA doubling occurs during early embryogenesis in T. fournieri and A. thaliana, indicating that the dynamics of mtDNA in early plant embryos differs from that in early mammalian embryos.     

Changes in the mitochondrial proteome of developing maize seed embryos

Mitochondria are required for seed development, but little information is available about their function and role during this process. We isolated the mitochondria from developing maize (Zea mays L. cv. Nongda 108) embryos and investigated the mitochondrial membrane integrity and respiration as well as the mitochondrial proteome using two proteomic methods, the two‐dimensional gel electrophoresis (2‐DE) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH). Mitochondrial membrane integrity and respiration were maintained at a high level up to 21 days after pollination (DAP) and decreased thereafter, while total mitochondrial number, cytochrome c oxidase activity and respiration per embryo exhibited a bell‐shaped change with peaks at 35–45 DAP. A total of 286 mitochondrial proteins changed in abundance during embryo development. During early stages of seed development (up to 21 DAP), proteins involved in energy production, basic metabolism, protein import and folding as well as removal of reactive oxygen species dominated, while during mid or late stages (35–70 DAP), some stress‐ and detoxification‐related proteins increased in abundance. Our study, for the first time, depicted a relatively comprehensive map of energy production by mitochondria during embryo development. The results revealed that mitochondria were very active during the early stages of maize embryo development, while at the late stages of development, the mitochondria became more quiescent, but well‐protected, presumably to ensure that the embryo passes through maturation, drying and long‐term storage. These results advance our understanding of seed development at the organelle level.     

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0–32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.     

Nip2/centrobin may be a substrate of Nek2 that is required for proper spindle assembly during mitosis in early mouse embryos

Nek2 is a mitotic kinase with multiple cellular functions involving phosphorylation of diverse substrates. Suppression of Nek2 in early mouse embryos has been shown to arrest development at the 4‐cell stage with defects in mitotic spindle assembly as well as in interphase nuclear morphology. In the present study, we suppressed expression of two Nek2 centrosomal substrates, Nip2 and C‐Nap1, in early mouse embryos. The development of the Nip2‐suppressed embryo was arrested at the 4‐cell stage with mitotic defects in the blastomeres. In contrast, C‐Nap1 suppression did not produce a visible phenotype. The phenotypic similarities of the Nip2‐ and Nek2‐suppressed embryos suggest that Nip2 may be a substrate of Nek2 that is required for mitotic spindle assembly in early mouse embryos. Mol. Reprod. Dev. 76: 587–592, 2009. © 2008 Wiley‐Liss, Inc.     

EMB1211 is required for normal embryo development and influences chloroplast biogenesis in Arabidopsis

Chloroplast biogenesis is tightly linked with embryogenesis and seedling development. A growing body of work has been done on the molecular mechanisms underlying chloroplast development; however, the molecular components involved in chloroplast biogenesis during embryogenesis remain largely uncharacterized. In this paper, we show that an Arabidopsis mutant carrying a T‐DNA insertion in a gene encoding a multiple membrane occupation and recognition nexus (MORN)‐containing protein exhibits severe defects during embryogenesis, producing abnormal embryos and thereby leading to a lethality of young seedlings. Genetic and microscopic studies reveal that the mutation is allelic to a previously designated Arabidopsis embryo‐defective 1211 mutant (emb1211). The emb1211 +/? mutant plants produce approximately 25% of white‐colored ovules with abnormal embryos since late globular stage when primary chloroplast biogenesis takes place, while the wild‐type plants produce all green ovules. Transmission electron microscopic analysis reveals the absence of normal chloroplast development, both in the mutant embryos and in the mutant seedlings, that contributes to the albinism. The EMB1211 gene is preferentially expressed in developing embryos as revealed in the EMB1211::GUS transgenic plants. Taken together, the data indicate that EMB1211 has an important role during embryogenesis and chloroplast biogenesis in Arabidopsis.     

Circadian genes and lithium response in bipolar disorders: associations with PPARGC1A (PGC‐1α) and RORA

Preliminary studies suggest that lithium (Li) response might be associated with some circadian gene polymorphisms, we therefore performed a pharmacogenetic study on the core clock genes in two independent samples suffering from bipolar disorder (BD) and thoroughly characterized for their Li response. Two independent Caucasian samples (165 and 58 bipolar patients) treated with Li were selected from samples recruited in a French multicenter study and assessed for their Li response using the Alda scale. The two samples were genotyped using the Human660 (H660) and OmniExpress (OE) BeadChips and gene‐based association analyses of 22 core clock genes were conducted. In the first sample (H660 chip), the RAR‐related orphan receptor‐a gene (RORA) and the Peroxisome Proliferator‐Activated Receptor Gamma, Coactivator 1 Alpha gene (PPARGC1A or PGC‐1α) were significantly associated with the Li response (empirical P‐value = 0.0015 and 0.04, respectively), and remained significant only for RORA after Bonferroni correction. In the second sample (OE chip), PPARGC1A was significantly associated with the Li response (empirical P‐value = 0.04), and did not remain significant after Bonferroni correction. PPARGC1A is a master regulator of mitochondrial function and a key component of the endogenous clock that stimulates the expression of Bmal1 and Rev‐erb‐alpha through coactivation of RORA. Although the observed associations deserve further replication and investigation, our results suggest genetic associations between Li response and these two close biological partners: PPARGC1A and RORA involved in circadian rhythms and bioenergetics processes in Li response.     

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.     

Mitochondrial ribosomal protein S9M is involved in male gametogenesis and seed development in Arabidopsis

• Mitochondrial function is critical for cell vitality in all eukaryotes including plants. Although plant mitochondria contain many proteins, few have been studied in the context of plant development and physiology.
• We used knock‐down mutant RPS9M to study its important role in male gametogenesis and seed development in Arabidopsis thaliana.
• Knock‐down of RPS9M in the rps9m‐3 mutant led to abnormal pollen development and impaired pollen tube growth. In addition, both embryo and endosperm development were affected. Phenotype analysis revealed that the rps9m‐3 mutant contained a lower amount of endosperm and nuclear proteins, and both embryo cell division and embryo pattern were affected, resulting in an abnormal and defective embryo. Lowering the level of RPS9M in rps9m‐3 affects mitochondrial ribosome biogenesis, energy metabolism and production of ROS.
• Our data revealed that RPS9M plays important roles in normal gametophyte development and seed formation, possibly by sustaining mitochondrial function.

     

Fatty acid synthesis in chick embryonic heart and liver during development.

Fatty acid synthesis by subcellular fractions of heart and liver of chick embryos at varying stages of development has been studied. Fatty acid synthetase activity is associated with the embryonic heart at early stages of development, as suggested by substrate requirement, Schmidt decarboxylation of synthesized fatty acids and gas liquid chromatographic identification of the products as palmitic and stearic acids. The fatty acid synthetase activity decreases in heart cytosol with age of the embryo and is absent in the newly hatched chick and in older chicken. The acetyl CoA carboxylase activity is negligible in embryonic and adult chicken heart. The fatty acid synthetase activity in liver is low, but measurable during the entire embryonic development. The activity increases by about three-fold on hatching and thereafter in fed, newly hatched chicks by about 35-fold, over the basal embryonic activity. The acetyl and malonyl transacylase activities in the heart and liver cytosols during development followed closely the fatty acid synthetase activities in heart and liver, respectively. A non-coordinate induction of fatty acid synthetase and acetyl CoA carboxylase activities in liver was observed during development. The microsomal chain elongation in liver and heart followed the pattern of fatty acid synthetase activity in liver and heart, respectively. The mitochondrial chain elongation in embryonic heart is initially low and increases with age; while this activity in liver is higher in early stages of embryonic development than in the older embryos and the chicks. Measurement of lipogenesis from acetate-1-¹⁴C by liver and heart slices from chick embryos and newly hatched chicks support the conclusions reached in the studies with the subcellular fractions. The results obtained indicate that the major system of fatty acid synthesis in embryonic and adult heart is the mitochondrial chain elongation. In embryonic liver, fatty acid synthesis proceeds by chain elongation, while the de novo system is the major contributor to the lipogenic capacity of the liver after hatching.     

Lipid interactions with in vitro development of mammalian zygotes

Rabbit zygotes were tested for their ability to sequester radiolabeled acetate, oleate, and arachidonate in intracellular lipid. Radiolabeled arachidonic acid was concentrated 170 ± 28-fold (mean ± SEM) and oleic acid was concentrated 105 ± 26-fold in zygotic lipids during 6 hr of culture when compared with the initial concentrations in culture medium. Acetate was not concentrated into lipids by cultured zygotes. Both long chain fatty acids were incorporated mainly as triglyceride. Polydimethylsiloxane fluid, used to cover the microdroplets of medium during culture, demonstrated lipophilic properties. This characteristic was utilized to indirectly transfer lipids to culture medium, permitting examination only of lipoidal properties of test extracts on embryonal development. For rabbit zygotes, blood plasma extract was detrimental and whole blood extract was beneficial for embryonal cleavage rates during the first 24 hr of culture. A higher proportion of mouse zygotes developed to blastocysts when cultured in modified Ham's F-10 medium compared to BMOC medium, and this difference was negated by inclusion of a lipid extract prepared from rabbit oviductal fluid in the culture system. Comparison of fatty acid analyses of the lipid extracts with development rates of zygotes suggests that modified rates of embryo development may be associated with ratios of individual fatty acids presented to the culture medium rather than with the presence of any single fatty acid.     

Correlation between porcinePPARGC1A mRNA expression and its downstream target genes in backfat andlongissimus dorsi muscle

Knowledge of in vivo relationship between the coactivatorPPARGC1A and its target genes is very limited, especially in the pig. In this study, a real-time PCR experiment was performed onlongissimus dorsi muscle (MLD) and backfat with 10 presumedPPARGC1A downstream target genes, involved in energy and fat metabolism, to identify possible relationships withPPARGC1A mRNA expression in vivo in the pig (n = 20). Except forUCP3 andLPL, a very significant difference in expression was found between MLD and backfat for all genes (P < 0.01). Hierarchical cluster analysis and the significant pairing of mRNA expression data between sampling locations suggested a genetic regulation of the expression of several target genes. A positive correlation withPPARGC1A was found forCPT1B, GLUT4, PDK4, andTFAM (P < 0.0001). A negative correlation was found forUCP2, FABP4, LEP (P < 0.0001), andTNF (P = 0.0071). No significant correlation was detected forUCP3 andLPL. This study provides evidence for a clear difference in mRNA expression of crucial genes in fat and energy metabolism between 2 important tissues. Our data suggest a clear impact ofPPARGC1A on energy and lipid metabolism in vivo in the pig, through several of these downstream target genes.     

Retinoic acid is a negative physiological regulator of N‐cadherin during early avian heart morphogenesis

The vitamin A‐deficient (VAD) early avian embryo has a grossly abnormal cardiovascular system that is rescued by treating the embryo with the vitamin A‐active form, retinoic acid (RA). Here we examine the role of N‐cadherin (N‐cad) in RA‐regulated early cardiovascular morphogenesis. N‐cad mRNA and protein are expressed globally in the presomite through HH14 normal and VAD quail embryos. The expression in VAD embryos prior to HH10 is significantly higher than that in normal embryos. Functional analyses of the N‐cad overproducing VAD embryos reveal N‐cad involvement in the RA‐regulated cardiovascular development and suggest that N‐cad expression may be mediated by Msx1. We provide evidence that in the early avian embryo, endogenous RA is a negative physiological regulator of N‐cad. We hypothesize that a critical endogenous level of N‐cad is needed for normal early cardiovascular morphogenesis to occur and that this level is ensured by stage‐specific, developmentally regulated RA signaling.