Glucose metabolism during bovine preimplantation development: analysis of gene expression in single oocytes and embryos.

peer reviewedGlucose 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

A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA.

The term "DNA fingerprint" has been used to describe the extensive restriction fragment length polymorphism associated with hypervariable minisatellites present in the human genome. Until now, it was necessary to hybridize Southern blots to specific probes cloned from human genomic DNA in order to obtain individual-specific restriction patterns. The present study describes the surprising finding that the insert-free, wild-type M13 bacteriophage detects hypervariable minisatellites in human and in animal DNA, provided no competitor DNA is used during hybridization. The effective sequence in M13 was traced to two clusters of 15-base pair repeats within the protein III gene of the bacteriophage. This unexpected use of M13 renders the DNA fingerprinting technology more readily available to molecular biology laboratories

Expression of Cu/Zn and Mn superoxide dismutases during bovine embryo development: influence of in vitro culture.

peer reviewedTemporal pattern of expression of Cu/Zn and Mn superoxide dismutases (SODs) was investigated in bovine oocytes and embryos produced in vitro in two different culture conditions and in vivo after superovulation. SODs were examined at a transcriptional level in single oocytes and embryos by reverse transcriptase-polymerase chain reaction (RT-PCR) and, at a protein level, by Western blotting on pools of embryos. mRNA encoding Cu/Zn SOD were detected in in vitro bovine embryos throughout preattachment development as well as in in vivo derived morulae and blastocysts. Transcripts for Mn SOD gene were detected in most immature and in vitro matured oocytes as well as in some zygotes and 5- to 8-cell embryos while no transcript was found at the 9-to 16-cell stage in both culture conditions. In vitro embryonic expression of Mn SOD was detected earlier in the presence of serum. Half of the morulae showed the transcript if cultured with 5% serum while none without serum. At the blastocyst stage Mn SOD could be detected independently of culture conditions. For in vivo-derived embryos Mn SOD transcripts were detected both in morulae and blastocysts. Immunoblotting analyses revealed that Cu/Zn SOD and Mn SOD were also present at a protein level in in vitro-derived zygotes and blastocysts. Together these data demonstrate, for the first time, that Mn SOD is transcribed and that Cu/Zn and Mn SOD proteins are expressed in preimplantation bovine embryos. Finally, they suggest that Mn SOD transcription is altered by in vitro culture conditions

How the new EU Dual-Use Regulation could benefit third countries?

peer reviewedEuropean Union P2P Programme on Export Control of Dual-use Goods Export Control - Chaudfontaine Grou

Transcription et stabilité des ARN messagers dans l'ovocyte et l'embryon bovin : interaction avec la cinétique de développement et les conditions de culture in vitro

Doctorat en sciences - UCL, 200

Poly(A) RNA is reduced by half during bovine oocyte maturation but increases when meiotic arrest is maintained with CDK inhibitors.

Variations in the amount of different RNA species were investigated during in vitro maturation of bovine oocytes. Total RNA content was estimated to be 2 ng before meiosis, and after meiosis resumption, no decrease was observed. Ribosomal RNA did not appear to be degraded either, whereas poly(A) RNA was reduced by half after meiosis resumption, from 53 pg to 25 pg per oocyte. Real-time polymerase chain reaction was performed on growth and differentiation factor-9 (GDF-9), on cyclin B1, and on two genes implicated in the resistance to oxidative stress, glucose-6-phosphate-dehydrogenase (G6PD) and peroxiredoxin-6 (PRDX6). When these transcripts were reverse-transcribed with hexamers, the amplification results were not different before or after in vitro maturation. But when reverse transcription was performed with oligo(dT), amplification was dramatically reduced after maturation, except for cyclin B1 mRNA, implying deadenylation without degradation of three transcripts. Although calf oocytes have a lower developmental competence, their poly(A) RNA contents were not different from that of cow oocytes, nor were they differently affected during maturation. When bovine oocytes were maintained in vitro under meiotic arrest with CDK inhibitors, their poly(A) RNA amount increased, but this rise did not change the poly(A) RNA level once maturation was achieved. The increase could not be observed under transcription inhibition and, when impeding transcription and adenylation, the poly(A) RNA decreased to a level normally observed after maturation, in spite of the maintenance of meiotic arrest. These results demonstrate the importance of adenylation and deadenylation processes during in vitro maturation of bovine oocytes

Impact of pro-oxidant agents on the morula-blastocyst transition in bovine embryos.

Exposing day 5 bovine morulae to reactive oxygen species induces a delayed degeneration of some blastocysts on day 8 post-insemination (pi) but without affecting the blastocyst rates. The aim of this study was to characterize the resisting and the degenerating population of blastocysts. The kinetics of degeneration of the embryos exposed to the two pro-oxidant agents: 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and buthionine sulfoximine (BSO) was evaluated using time-lapse cinematography. With both agents the first signs of degeneration appeared at day 7.5 pi but the duration of the degeneration process was shorter in presence of AAPH than BSO (4.2 vs. 12.5 hr, ANOVA, P < 0.05). The resisting blastocysts derived from morulae with a larger diameter (mean diameter: 161 vs. 154 microm, ANOVA, P < 0.05) and showed an earlier cavitation (135 vs. 142 hpi, P < 0.05) than the degenerating ones. The profile of protein neosynthesis at day 7 was not affected by the treatment. The proportion of male embryos was more important in the resisting than in the degenerating population (70 vs. 55%, chi2, P < 0.05) especially when the stress was induced by AAPH. The quality of the resisting embryos, measured by the total cell number and the rate of apoptosis, did not seem to be affected when compared to control embryos. In conclusion, resistance to oxidative stress seems related to the kinetics of development and/or the sex of the embryos. Resisting embryos apparently display a quality similar to untreated embryos

Effects of stress on blood pressure measured by Doppler ultrasonography and high definition oscillometry in a large population of working dogs

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Effect of stress on blood pressure measured by Doppler ultrasonography and high definition oscillometry in a large population of working dogs

The stress induced by the indirect blood pressure measurement procedure may contribute to increase systolic blood pressure and sometimes lead to a false diagnosis of hypertension. Cortisol dosage has already been used as an indicator of stress state in dogs. The purpose of this study was to compare, in a well standardized population of working dogs, the influence of stress on systolic blood pressure measured by two indirect blood pressure devices: Doppler ultrasonography and high definition oscillometry (HDO). This study is included in the lupa project, a large European collaboration aiming at the identification of genetic variants influencing blood pressure. First, we tested the intra-day (5 measures) and inter-day (5 exams) repeatability of the two devices in 5 beagle dogs. The variation coefficients for systolic arterial pressure (SAP) measured by Doppler and HDO were lower than 10%. Thereafter, we included 70 healthy Belgian Shepherds from Belgian police department and from Belgian army. All dogs were male, working dogs, between 2 and 6 years old. The SAP was always measured by the same trained operator, under the same standardized procedure, in a calm environment. The Doppler measurement was made at the beginning of the exam and the HDO measurement 20 minutes after the Doppler test. At the end of the exam, a blood sample was taken to measure cortisol level. At each measurement, stress level and difficulty to perform the exam were scored by a four levels scale. The SAP measured by Doppler and HDO were highly correlated (R = 0.429, p < 0,001). Nevertheless, SAP measured by the Doppler device (159mmHg ± 3.374) was higher (p < 0,001.) than SAP measured by HDO (143mmHg ± 1.877). The SAP measured by the Doppler device was positively correlated to the stress level (R = 0.612, p < 0,001), the difficulty of measurement (R = 0.394, p < 0.01) and blood cortisol concentration (R = 0.336, p < 0.02) while only a weak correlation was observed between SAP measured by HDO and the stress level (R = 0,262, p = 0,045). These results suggest that the SAP value measured by a Doppler device is more importantly influenced by the stress level than the SAP value measured by HDO. Therefore, in nervous dogs such as working Belgian Shepherds, the HDO method seems more suitable than the Doppler method