Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo

This study was an investigation of metabolism during bovine preimplantation development from the oocyte up to the hatched blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose, pyruvate, or lactate during a 4-h incubation period in a closed noninvasive system with NaOH as trap for the continuous collection of CO(2). The postincubation medium was analyzed for the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with pyruvate being the preferred substrate. The first marked increase in oxidation of glucose occurred between the 12- and 16-cell stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily 15-fold from the 1-cell to the blastocyst stage. In general, the pattern of metabolism was similar between the embryos derived in vitro and in vivo but with some distinct differences. The most apparent feature of glucose metabolism by in vitro-produced embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivo-matured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production increased significantly and at the hatched blastocyst stage matched that of their in vitro counterparts. In vitro-produced embryos up to the 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to-lactate oxidation than the in vivo-obtained embryos. The results of this study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos produced in vitro and those generated in vivo that may well affect the developmental capacity.     

In vitro development of reconstructed bovine embryos and fate of donor mitochondria following nuclear injection of cumulus cells

In this study we examined the developmental potential of reconstructed embryos and the fate of donor mitochondria during preimplantation development after nuclear transfer in cattle. Isolated cumulus cells were used as donor cells in nuclear transfer. Cumulus cells labelled with MitoTracker Green FM fluorochrome were injected into enucleated bovine MII oocytes and cultured in vitro. MitoTracker labelling on donor cells did not have a detrimental effect on blastocyst formation following nuclear transfer. Cleavage rate was about 69% (56/81) and blastocyst formation rate was 6.2% (5/81) at 7 days after nuclear transfer. The labelled mitochondria dispersed to the cytoplasm and became distributed between blastomeres and could be identified up to the 8- to 15-cell stage. Small patches of mitochondria were detected in some 8- to 15-cell stage embryos (5/20). However, donor mitochondria were not detected in embryos at the 16-cell stage and subsequent developmental stages. In the control group, mitochondria could be identified in arrested 1-cell embryos up to 7 days after nuclear transfer. These results suggest that disappearance of the labelled donor mitochondria in nuclear transfer bovine embryos is not due to fading of the fluorochrome marker, but is rather an as yet undefined cytoplasmic event.     

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.     

The development of preimplantation mouse parthenogenones in vitro in absence of glucose: Influence of the maternally inherited components

Mouse preimplantation embryo development is characterized by a switch from a dependence on the tricarboxylic acid cycle pre-compaction to a metabolism based on glycolysis post-compaction. In view of this, the role of glucose in embryo culture medium has come under increased analysis and has lead to improved development of outbred mouse embryos in glucose free medium. Another type of embryo that has proven difficult to culture is the parthenogenetic (PN) mouse embryo. With this in mind we have investigated the effect of glucose deprivation on PN embryo development in vitro. Haploid and diploid PN embryos were grown in medium M16 with or without glucose (M16-G) and development, glycolytic rate, and methionine incorporation rates assessed. Haploid PN and normal embryo development to the blastocyst stage did not differ in either M16 or M16-G. In contrast, although diploid PN embryos formed blastocysts in M16 (28.3%), they had difficulty in undergoing the morula/blastocyst transition in M16-G (7.6%). There was no significant difference in mean cell numbers of haploid PN, diploid PN and normal embryos cultured in M16 and M16-G at the morula and blastocyst stage. Transfer of diploid PN embryos from M16-G to M16 at the four- to eight-cell stage dramatically increased blastocyst development. At the morula stage diploid PN embryos grown in M16-G exhibited a higher glucose metabolism and protein synthesis compared to those grown in M16 and to haploid PN embryos. Difficulties of diploid PN embryos in undergoing the morula/blastocyst transition in absence of glucose infer the existence of a link between the maternally inherited components and the preimplantation embryos dependence on glucose. © 1996 Wiley-Liss, Inc.     

Intracytoplasmic glutathione concentration and the role of beta-mercaptoethanol in preimplantation development of bovine embryos

In vitro-matured/in vitro-fertilized bovine oocytes were cultured on cumulus cell layers in a serum-free medium (bovine embryo culture medium; BECM) supplemented with 3 mg/ml fatty acid-free BSA. The intracytoplasmic glutathione concentration of embryos was found to change significantly (P < 0.008) during the preimplantation stages, beginning to increase at the 9- to 16-cell stage (20.7 pM/embryo) and reaching the highest (P < 0.03) level at the hatched-blastocyst stage (36.7 pM/embryo). A significantly (P < 0.06) lower concentration of glutathione was obtained at the 2- to 8-cell stage (7.1 pM/embryo) than at any other stage. When inseminated oocytes were cultured in BECM supplemented with different concentrations of beta-mercaptoethanol (2-ME) to promote glutathione synthesis, higher (P < 0.05) percentages of embryos developed to the 9- to 16-cell, morula and blastocyst stages at 96, 144 and 192 h post insemination, following the addition of 6.25 and 12.5 microM than after no supplementation with 2-ME. However, when 16-cell embryos were cultured in BECM supplemented with 6.25 and 12.5 microM of 2-ME, blastocyst formation was not significantly (P > 0.9) increased. When the combined effects of 2-ME and/or cumulus cells were compared in a 2 x 2 factorial design, there was a significant (P < 0.03) effect of 2-ME on the development of oocytes to blastocysts. The presence of cumulus cells significantly (P < 0.001) affected development after the fourth cleavage (morula compaction and blastocyst formation), but there was no significant (P > 0.11) interaction between 2-ME and cumulus cells. In conclusion, intracytoplasmic glutathione concentration of bovine embryos derived from in vitro-culture increases during preimplantation development. The glutathione synthesis promoter 2-ME exerts its embryotropic role on the development before the fourth cleavage, thus yielding an improvement in blastocyst formation.     

U2 small nuclear RNA localization and expression during bovine preimplantation development.

This study describes the localization of the U2 small nuclear RNA (snRNA) and the major U snRNA group ribonucleoproteins (snRNPs) during bovine preimplantation development. In vitro maturation, fertilization, and oviductal epithelial cell coculture methods were employed to produce several developmental series totalling over 2,000 preimplantation-stage bovine oocytes and embryos. These oocytes and preimplantation embryos were processed for in situ hybridization, immunofluorescence and Northern blotting methods. The U2 snRNA and the major U group snRNPS were localized initially over the germinal vesicle (GV) of preovulatory oocytes but following GV breakdown were released throughout the ooplasm. They subsequently reassociated with both pronuclei during fertilization. From the two-cell to the blastocyst stages, the U2 snRNA and U snRNPs were localized to the interphase nucleus of each blastomere. The levels of U2 snRNA throughout bovine preimplantation development were determined by probing a Northern blot containing total RNA isolated from the following preimplantation bovine embryo stages: one to two cell, eight to 16 cell, early morula (greater than 32 cell), and late morula/early blastocysts. The levels of U2 snRNA remained constant between the one-cell and eight- to 16-cell bovine embryo stages but increased 4.4-fold between the eight- to 16-cell stage and the late morula/early blastocyst stages. The results suggest that a maternal pool of snRNAs is maintained in mammalian preimplantation embryos regardless of the duration of maternal control of development.     

Glucose transporter gene expression in early mouse embryos.

The glucose transporter (GLUT) isoforms responsible for glucose uptake in early mouse embryos have been identified. GLUT 1, the isoform present in nearly every tissue examined including adult brain and erythrocytes, is expressed throughout preimplantation development. GLUT 2, which is normally present in adult liver, kidney, intestine and pancreatic beta cells is expressed from the 8-cell stage onward. GLUT 4, an insulin-recruitable isoform, which is expressed in adult fat and muscle, is not expressed at any stage of preimplantation development or in early postimplantation stage embryos. Genetic mapping studies of glucose transporters in the mouse show that Glut-1 is located on chromosome 4, Glut-2 on chromosome 3, Glut-3 on chromosome 6, and Glut-4 on chromosome 11.     

Role of glucose in mouse preimplantation embryo development

Mouse preimplantation embryos consume pyruvate preferentially during the early developmental stages, before glucose becomes the predominant energy substrate in the blastocyst. To investigate the importance of the switch to glucose utilization at the later developmental stages, mouse embryos from F1 hybrid mice (CBA/Ca × C57BL/6) were cultured from the one-and two-cell stages (22 and 46 h post hCG, respectively) for 5 days in a modified medium, M16, containing 0.33 mM pyruvate and 5 or 23 mM D+L-lactate, in the presence and absence of 1 mM glucose (M16+G and M16-G, respectively). Nutrient uptakes were also determined over this time. Some embryos cultured in M16-G were transferred to M16+G at 94 or 118 h post hCG. Embryos cultured from the two-cell stage in M16+G exhibited the characteristic fall in pyruvate consumption between the morula and the blastocyst stage; those cultured from the two-cell stage in M16-G compensated for the lack of glucose by consuming increasing amounts of pyruvate, from 2.78 pmol/embryo/h at 58 h post hCG to 5.21 pmol/embryo/h at 154 h post hCG. However, the percentage of embryos developing to the blastocyst stage, the hatching rate, and blastocyst cell numbers (50.6 ± 2.5 [28] vs. 105 ± 3.8 [37]) were all lower in this group. When exposed to glucose at 94 or 118 h post hCG, embryos cultured from the two-cell stage in M16-G readily consumed glucose in preference to pyruvate, although the characteristic fall in pyruvate consumption was not observed. One-cell embryos cultured continuously in M16-G were only able to develop to the morula stage, after which time they degenerated. In these embryos pyruvate was readily consumed between 22 and 94 h post hCG, before falling from 2.77 pmol/embryo/h at 83 h post hCG to 0.045 pmol/embryo/h at 130 h post hCG. Transfer of these embryos to M16+G at 94 and 118 h post hCG did not support development to the hatching blastocyst stage. The results show that mouse preimplantation embryos from F1 hybrid mice (CBA/Ca × C57BL/6) need only be exposed to glucose for less than 24 h between 22 and 94 h post hCG in order to develop from the morula to the blastocyst stage in vitro. However, the exposure time needs to be increased to between 24 and 72 h in order that blastocyst cell numbers reach control levels. The importance of glucose before the morula stage may relate to the need to synthesize glycogen for later use. If the obligatory requirement for glucose is fulfilled, embryos are able to utilize pyruvate in the absence of glucose at the later stages of development. These results show that the mouse preimplantation embryo can, to some extent, adapt metabolically to changes in its external environment. © 1995 Wiley-Liss, Inc.     

Decreased glucose transporter expression triggers BAX-dependent apoptosis in the murine blastocyst

We report that a decrease in facilitative glucose transporter (GLUT1) expression and reduced glucose transport trigger apoptosis in the murine blastocyst. Inhibition of GLUT1 expression either by high glucose conditions or with antisense oligodeoxynucleotides significantly lowers protein expression and function of GLUT1 and as a result induces a high rate of apoptosis at the blastocyst stage. Similar to wild-type mice, embryos from streptozotocin-induced diabetic Bax -/- mice experienced a significant decrease in glucose transport compared with embryos from non-diabetic Bax -/- mice. However, despite this decrease, these blastocysts demonstrate significantly fewer apoptotic nuclei as compared with blastocysts from hyperglycemic wild-type mice. This decrease in preimplantation apoptosis correlates with a decrease in resorptions and malformations among the infants of the hyperglycemic Bax -/- mice versus the Bax +/+ and +/- mice. These findings suggest that hyperglycemia by decreasing glucose transport acts as a cell death signal to trigger a BAX-dependent apoptotic cascade in the murine blastocyst. This work also supports the hypothesis that increased apoptosis at a blastocyst stage because of maternal hyperglycemia may result in loss of key progenitor cells and manifest as a resorption or malformation, two adverse pregnancy outcomes more common in diabetic women.