Anne-Sophie Lequarre

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

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‐phosphate‐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 mRNA 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. Mol. Reprod. Dev. 48:216–226, 1997.

Cell Cycle Duration at the Time of Maternal Zygotic Transition for In Vitro Produced Bovine Embryos: Effect of Oxygen Tension and Transcription Inhibition

Abstract Early embryonic cleavages are mostly regulated by maternal components then control of development progressively depends on newly synthesized zygotic products. The timing of the first cleavages is a way to assess embryo quality. The goal of this study was to evaluate the duration of the fourth cell cycle, at the time of maternal-to-zygotic transition (MZT) in in vitro-produced bovine embryos by means of cinematographic analysis. We found that 75% of the embryos displayed a long fourth cycle (43.5 ± 5.4 h) whereas the remaining embryos had a very short fourth cell cycle (8.9 ± 2.9 h). Both groups did not differ in cleavage rhythm up to the eight-cell stage and timing of cavitation and blastocyst expansion was identical. However, embryos with a short fourth cell cycle had a better blastocyst rate than embryos with a long cycle (59% versus 38%, P < 0.01). Total cell number, inner cell mass (ICM):total cell ratio, and hatching rate were identical for blastocysts produced from embryos with either a long or a short fourth cell cycle. In a second experiment, we showed that increasing the oxygen tension, from 5% to 20%, decreased the percentage of embryos with a short fourth cell cycle, from 25% to 11% (P < 0.01), indicating that suboptimal culture conditions can influence the length of this cycle. Finally, we investigated whether fourth cell cycle duration could be influenced by transcription inhibition. With alpha-amanitin added at 18 h postinsemination (HPI), cleavage was reduced (66% versus 79%) and, at 70 HPI, the 9- to 16-cell rate increased (50% versus 25%) concomitantly with a 5- to 8-cell rate decrease (16% versus 47%). A similar pattern was observed when the drug was added at 6 HPI or 42 HPI but not at 0 HPI. Cinematographic analysis revealed that alpha-amanitin increased the first cell cycle duration whereas the second and third cell cycles were not affected. With the drug, one third of the embryos could develop up to the 9- to 16-cell stage and they all had a short fourth cell cycle (11.2 ± 3.7 h) with a good synchrony of cleavage between blastomeres. These results suggest that duration of the fourth cell cycle of bovine embryo, during the MZT, is under a zygotic transcriptional control that can be affected by oxidative conditions.

Poly(A) RNA Is Reduced by Half During Bovine Oocyte Maturation but Increases when Meiotic Arrest Is Maintained with CDK Inhibitors

Abstract 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.

EXPRESSION OF CU/ZN AND MN SUPEROXIDE DISMUTASES DURING BOVINE EMBRYO DEVELOPMENT: INFLUENCE OF IN VITRO CULTURE

Temporal 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. Mol. Reprod. Dev. 58:45–53, 2001.

Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism.

Embryos derived from calf oocytes were compared with adult cow oocyte‐derived embryos (1) by studying the kinetics of embryo development using time‐lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte‐derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte‐derived embryos preferably arrested their development before the 9‐cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2‐cell or 3‐cell stages than embryos that arrested before the 9‐cell stage. The 9‐cell stage tended to appear later in calf oocyte‐derived embryo that reached the blastocyst stage than in adult‐derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte‐derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89u2009±u200920; cow: 100u2009±u200930) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64u2009±u200915 vs. 65u2009±u20096u2009ng/embryo, respectively). In conclusion, calf oocyte‐derived embryos are characterized by a higher rate of developmental arrest before the 9‐cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient “capacitation” of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected. Mol. Reprod. Dev. 57:346–352, 2000.