ABSTRACT
Glucose metabolism of the bovine embryo is low during the first cleavages and increases sharply after the major resumption of the genome (8-16 cells). The mRNA level for genes involved in glucose metabolism was tested by RT-PCR on individual oocytes and embryos at different stages of development. These genes were: glucose transport GLUT-1, hexokinase (HK), glucose-6-phosphatase-dehydrogenase (G6PDH), and glucose-phosphate-isomerase (GPI); actin was used as a reference transcript. RT-PCR results revealed three types of oocytes or embryos: positive with a PCR signal for each transcript considered, nul with no signal for any transcript, and heterogeneous with a PCR signal for some transcripts and none for others. The number of nul and heterogeneous samples was higher for slow than for fast-cleaving embryos (81% vs. 36%), and the proportion of positive embryos increased significantly at the 16-cell and morula stages (P < 0.002), suggesting a correlation between mRNA content and developmental capacity. In positive embryos, GLUT-1 level was reduced by half during maturation and fertilization. Actin and hexokinase mRNA levels decreased during the first cleavages, but significantly increased at the 16-cell and morula stages, respectively. GPI transcript remained stable throughout development, whereas there was a significant rise for G6PDH at the 4-cell stage, perhaps due to a polyadenylation process. Finally, the absence or decrease in intensity of several transcripts at the blastocyst stage suggests suboptimal culture conditions.
Subject(s)
Embryo, Mammalian/metabolism , Embryonic Development , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Glucose/metabolism , Oocytes/metabolism , Actins/genetics , Animals , Cattle , Female , Glucose Transporter Type 1 , Glucose-6-Phosphate Isomerase/genetics , Glucosephosphate Dehydrogenase/genetics , Hexokinase/genetics , Monosaccharide Transport Proteins/genetics , Polymerase Chain Reaction , Pregnancy , Transcription, GeneticABSTRACT
The effects of fetal calf serum (FCS) or serum fractions on the development of bovine embryos was investigated. Bovine zygotes were produced in vitro and were cultured in a semi-defined culture medium (mSOF). In the first experiment, blastocysts produced in mSOF supplemented with 10% whole heat-treated FCS or desalted FCS appeared about 1 day earlier, their proportion was significantly (P < 0.05) higher (whole: 30%, desalted: 29%) and they had significantly (P < 0.05) more cells at day 8 (119 cells, 127 cells) than did blastocysts produced in mSOF without any supplement (16%, 98 cells) or mSOF supplemented with a glucose concentration equivalent to that of serum (15%, 88 cells). Our results indicate that high molecular mass components (> 5 kDa) of serum are responsible for the effects of FCS on the kinetics of development, on the percentage of blastocysts obtained and the total number of cells in blastocysts. A further analysis using time-lapse microcinematography showed that the acceleration of development induced by serum occurred between the 9-16-cell and morula stages. Finally, in an experiment designed to analyse by microcinematography the effect of the addition of FCS using semen from a different bull to inseminate the oocytes, a different batch of serum and adding mSOF at a different time (42 h after insemination), acceleration was similarly observed between these two stages. Our microcinematographic studies demonstrate that the addition of FCS at two developmental stages (three-four-cell and five-eight-cell) before the 8-16-cell stage accelerates development just after this critical blocking stage.
Subject(s)
Cell Culture Techniques/methods , Embryonic and Fetal Development , Fertilization in Vitro , Animals , Cattle , Culture Media , Female , Fetal Blood , Male , Time FactorsABSTRACT
The aim of this study was to try establishing mouse ES cell lines from the early developmental stage. Fifty-two uncompacted 8-cell stage embryos were dissociated and single blastomeres were seeded on primary embryonic fibroblasts in DMEM/F12 completed with 10% foetal calf serum, 10% new born calf serum. 10(-4) M beta-mercaptoethanol. After approximately 5 days of culture, multiple cell clones exhibiting stem cell morphology grew out and were dissociated. One cell line was established (MSB1) and characterised. The karyotype and the G-banding revealed a male diploid cell line. MSB1 cells were injected into syngenic mice and produced teratocarcinomas. Detailed histological examination of the tumours showed a great variety of cell types including representatives of all three primary germ layers. Several nests of undifferentiated stem cells were also present. Microinjections of MSB1 cells into 52 blastocysts produced 2 chimeras, 1 male and 1 female. These results demonstrate that a highly pluripotents ES cell line can be derived from 8-cell stage mouse embryos. However, the male chimera appeared sterile. More experiments would thus be necessary to prove that the cell line obtained is capable to colonise the germ line.