ascorbate peroxidase ͉ chloroplast ͉ oxidative stress ͉ reactive oxygen species

SummaryVitamin B6 is an essential coenzyme for numerous metabolic enzymes and is a potent antioxidant. In plants, very little is known about its contribution to viability, growth and development. The de novo pathway of vitamin B6 biosynthesis has only been described recently and involves the protein PDX1 (pyridoxal phosphate synthase protein). Arabidopsis thaliana has three homologs of PDX1, two of which, PDX1.1 and PDX1.3, have been demonstrated as functional in vitamin B6 biosynthesis in vitro and by yeast complementation. In this study, we show that the spatial and temporal expression patterns of PDX1.1 and PDX1.3, investigated at the transcript and protein level, largely overlap, but PDX1.3 is more abundant than PDX1.1. Development of single pdx1.1 and pdx1.3 mutants is partially affected, whereas disruption of both genes causes embryo lethality at the globular stage. Detailed examination of the single mutants, in addition to those that only have a single functional copy of either gene, indicates that although these genes are partially redundant in vitamin B6 synthesis, PDX1.3 is more requisite than PDX1.1. Developmental distinctions correlate with the vitamin B6 content. Furthermore, we provide evidence that in addition to being essential for plant growth and development, vitamin B6 also plays a role in stress tolerance and photoprotection of plants.

BackgroundPreimplantation bovine development is emerging as an attractive experimental model, yet little is known about the mechanisms underlying trophoblast (TE)/inner cell mass (ICM) segregation in cattle. To gain an insight into these processes we have studied protein and mRNA distribution during the crucial stages of bovine development. Protein distribution of lineage specific markers OCT4, NANOG, CDX2 were analysed in 5-cell, 8–16 cell, morula and blastocyst stage embryos. ICM/TE mRNA levels were compared in hatched blastocysts and included: OCT4, NANOG, FN-1, KLF4, c-MYC, REX1, CDX2, KRT-18 and GATA6.ResultsAt the mRNA level the observed distribution patterns agree with the mouse model. CDX2 and OCT4 proteins were first detected in 5-cell stage embryos. NANOG appeared at the morula stage and was located in the cytoplasm forming characteristic rings around the nuclei. Changes in sub-cellular localisation of OCT4, NANOG and CDX2 were noted from the 8–16 cell onwards. CDX2 initially co-localised with OCT4, but at the blastocyst stage a clear lineage segregation could be observed. Interestingly, we have observed in a small proportion of embryos (2%) that CDX2 immunolabelling overlapped with mitotic chromosomes.ConclusionsCell fate specification in cattle become evident earlier than presently anticipated – around the time of bovine embryonic genome activation. There is an intriguing possibility that for proper lineage determination certain transcription factors (such as CDX2) may need to occupy specific regions of chromatin prior to its activation in the interphase nucleus. Our observation suggests a possible role of CDX2 in the process of epigenetic regulation of embryonic cell fate.

The aim of the present study was to investigate whether protein or macromolecule supplements to in vitro maturation media affect transcript abundance of seven genes (Bax, Bcl2, Hsp70, IGF1, IGF1R, IGF2, and IGF2R) in oocytes and blastocysts. Cumulus-oocyte complexes aspirated from slaughterhouse ovaries were matured in TCM199 medium supplemented either with 10% FBS, 6% fatty acid free BSA (fafBSA) or 4% PVP40, then inseminated and cultured in vitro for 9 days. Transcript abundance analysis was carried out on immature and in vitro matured oocytes, as well as on blastocysts. Total RNA was isolated from pools of oocytes and embryos, reverse transcribed into cDNA and subjected to transcript analysis by real-time PCR. No transcript of IGF1 gene was detected either in oocytes or in blastocysts. Maturation conditions significantly affected transcript levels of investigated loci in blastocysts but not in matured oocytes, with one exception. Only relative abundance (RA) of IGF2 gene was higher in oocytes matured with fafBSA. Moreover, oocyte maturation with fafBSA elevated transcript abundance of IGF1R, IGF2, and IGF2R genes in resulting blastocysts, whereas Hsp70 transcription was stimulated by FBS supplementation. Thus, under described conditions, fafBSA may be the optimal supplement to IVM medium due to higher transcript level of growth factor coding genes accompanied by a lower transcript level of Hsp70.

Oocyte quality is affected by many factors, among which the environment of growth and maturation seems to be crucial. Studies show that well balanced oocyte energy metabolism has a significant impact on several elements of cytoplasmic and nuclear maturation as well as further embryo developmental competence. Therefore homeostasis between metabolism of glucose and fatty acids in the oocyte is being widely described nowadays. This review aims to discuss the follicular (in vivo) or maturation media (in vitro) environments with regard to glucose and fatty acid metabolism, as the main sources of the energy for the oocyte. A great emphasis is given on the balance between those two metabolic pathways and its further impact on female fertility.