Effect of amino acids and α-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES

Four experiments were conducted to test the effects of Eagle's non-essential amino acids (NEAA) and essential amino acids (EAA), glycine, and the RNA polymerase inhibitor α-amanitin, on the development of preimplantation rabbit embryos in modified protein-free KSOM medium. Embryos were distributed randomly into different treatments and cultured in 5% O₂:5% CO₂:90% N₂. In experiment 1, 100% of the embryos became blastocysts in the medium with Eagle's IX NEAA and 0.5X EAA, but 100% stopped development at the morula stage in KSOM without amino acids. These morulae failed to develop further when transferred to amino acid supplemented medium after 72 hr of culture. Glycine alone in modified KSOM (experiment 2) was ineffective in supporting development of 8–16-cell stage embryos past the morula stage. In experiment 3, the addition of IX NEAA and 0.5X EAA at 0, 12, 24, 36, and 48 hr of culture resulted, respectively, in 57, 65, 65, 44, and 14% blastocysts on Day 3 (P<0.05) and 86, 77, 77, 78, and 69% on Day 5 (P<0.05). Omission of Eagle's amino acids until 48 hr clearly delayed embryo development. In experiment 4, when α-amanitin (20 μM) was added to the medium containing Eagle's amino acids after 0, 12, 24, 36, and 48 hr of culture most embryos cleaved only once or twice after adding the α-amanitin. Without the inhibitor, 94% of the zygotes developed into blastocysts. These results indicate that modified KSOM or KSOM plus glycine could not support rabbit embryo development past the morula stage, but this block was overcome by adding Eagle's amino acids. An exogenous source of amino acids was not critical for embryo development during the first 24 hr of culture, but was required after that for development to equal controls. Addition of α-amanitin at multiple pre-blastocyst stages limited further embryo development to one or two cleavage divisions, with no blastocyst development. © 1996 Wiley-Liss, Inc.     

Evolutionary modification of specification for the endomesoderm in the direct developing echinoid <Emphasis Type="Italic">Peronella japonica</Emphasis>: loss of the endomesoderm-inducing signal originating from micromeres

We investigated the inductive signals originating from the vegetal blastomeres of embryos of the sand dollar Peronella japonica, which is the only direct developing echinoid species that forms micromeres. To investigate the inductive signals, three different kinds of experimental embryos were produced: micromere-less embryos, in which all micromeres were removed at the 16-cell stage; chimeric embryos produced by an animal cap (eight mesomeres) recombined with a micromere quartet isolated from a 16-cell stage embryo; and chimeric embryos produced by an animal cap recombined with a macromere-derived layer, the veg1 or veg2 layer, isolated from a 64-cell stage embryo. Novel findings obtained from this study of the development of these embryos are as follows. Micromeres lack signals for endomesoderm specification, but are the origin of a signal establishing the oral–aboral (O–Ab) axis. Some non-micromere blastomeres, as well as micromeres, have the potential to form larval skeletons. Macromere descendants have endomesoderm-inducing potential. Based on these results, we propose the following scenario for the first step in the evolution of direct development in echinoids: micromeres lost the ability to send a signal endomesoderm induction so that the archenteron was formed autonomously by macromere descendants. The micromeres retained the ability to form larval spicules and to establish the O–Ab axis.     

Co-culture of rabbit 2-cell embryos with rabbit oviduct epithelial cells and other somatic cells

Rabbit 2-cell embryos were co-cultured in Basel Synthetic Medium II + 10% fetal bovine serum with one of the following: primary cultures of rabbit oviduct epithelial cells (ROEC), a rabbit kidney epithelioid cell line (RK13), a rabbit epidermal epithelioid cell line (Sf1), or a rabbit skin fibroblast-like cell line (RAB9). Embryos cultured in medium alone served as controls. After 4 d of culture at 39 degrees C in 5% CO2 in air, 77-93% of the rabbit embryos which were co-cultured with somatic cells had reached the blastocyst stage, and 60-76% were hatching through their zonae pellucidae. These percentages, however, were not significantly different (P greater than .05) from those of embryos in medium alone, of which 90% had reached the blastocyst stage and 83% were hatching. Mean intrazonal embryo diameters also did not differ significantly among treatments (239-302 microns). Bovine 1-8-cell embryos were also co-cultured with ROEC. This stimulated 60% of these embryos to develop beyond the so-called "16-cell block" in vitro, whereas 0% of the embryos cultured in medium alone developed past this block. Evaluation of the ROEC cultures by light microscopy, immunocytochemistry, and gel electrophoretic analysis of conditioned medium, together with the positive results with bovine embryos, indicate that the ROEC culture partially simulates oviductal conditions in vivo. Therefore, our results suggest that oviduct epithelial cells may play a less pivotal role in regulating early development in the rabbit than in the cow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variations in the amounts of RNA polymerase forms I, II and III during preimplantation development in the mouse.

DNA-dependent RNA polymerase has been measured at various stages of preimplantation development in mouse embryos. The total RNA polymerase activity per embryo increases rapidly from the 8-cell stage to the blastocyst stage. Studies with low α-amanitin concentrations, which inhibit form II RNA polymerase, and high α-amanitin concentrations, which inhibit both form II and III RNA polymerases indicate that the relative proportions of the three forms change significantly during preimplantation development. The changes which occur in the types and levels of RNA polymerase appear to parallel corresponding changes in the synthesis of the major classes of RNA.     

Control of protein synthesis during blastocyst formation in the mouse.

In order to evaluate the dependence of the embryo on new mRNA synthesis during the period leading to blastulation, quantitative and qualitative aspects of protein synthesis in developing mouse morulae were investigated using α-amanitin, an inhibitor of RNA polymerase II. Only 1 of 423 early morulae cultured for 27 hr in the presence of 11 μg/ml α-amanitin cavitated, although most progressed as far as fully compacted morulae. About two-thirds of the untreated embryos cavitated during the same period. Incorporation of [³⁵S]methionine into protein was measured at 3- or 4-hr intervals over a 24-hr period and showed a two- to fivefold increase in control embryos. This increase was blocked in the α-amanitin-treated group although initial levels of incorporation were maintained. Total uptake of the amino acid appeared to be unaffected by the inhibitor. RNA synthesis, as measured by [³H]uridine incorporation over the same period, was reduced by between 5 and 52%, and the preblastulation surge in RNA synthesis was also blocked by α-amanitin. Two-dimensional polyacrylamide gel electrophoresis of labeled polypeptides synthesized by the embryos after 24-hr incubation in the presence or absence of the inhibitor revealed three distinct classes of polypeptide. The majority of polypeptides continued to be synthesized in the presence of α-amanitin whereas a small number of polypeptides, the synthesis of which would normally have increased during the development of the morula to the blastocyst, were prevented from doing so. A few polypeptides which normally cease to be synthesized over this period continued to be synthesized in the presence of α-amanitin. It is concluded that, while most of the proteins detectable at the morula stage are synthesized on mRNA templates of relatively long translational life, the general surge in protein synthesis, including the increased synthesis of a few species of polypeptide, are dependent on continuous translational activity.     

Developmental failure of hybrid embryos generated by in vitro fertilization of water buffalo (Bubalus bubalis) oocyte with bovine spermatozoa

The developmental potential of inter-species hybrid embryos produced by in vitro fertilization of in vitro matured buffalo oocytes with bovine spermatozoa was studied with a view to investigate pre-implantation embryo development and its gross morphology, early embryonic gene expression, and embryonic genome activation. Fertilization events with both buffalo and cattle spermatozoa were almost similar. Overall fertilization rate with cattle spermatozoa was 78.4% was not significantly different from that of buffalo spermatozoa (80.2%). Initial cleavage rate between buffalo and hybrid embryo was also similar, and there was no significant difference in their developmental rate till 8-cell stage (26.0 +/- 4.1 vs. 24.3 +/- 4.8). However, only 5.3% of hybrid embryos developed into blastocyst stage compared to 21.7% in buffalo. mRNA phenotyping of insulin-like growth factor family (Insulin, insulin receptor, IGF-I, IGF-I receptor, IGF-II, and IGF-II receptor) and glucose transporter isoforms (GLUT-I, II, III, IV) in hybrid embryos clearly showed that these molecules were not expressed after 8-cell stage onward. Similarly, as observed in buffalo embryos, incorporation of (35)S-methionine and (3)H-uridine could not be observed in hybrid embryos from 8-cell stage onward. This suggests that the maternal-zygotic genome activation did not occur in hybrid embryos. Differential staining also showed that the blastomere stopped dividing after 8-cell stage. Collectively, these parameters clearly showed that there was developmental failure of hybrid embryos.     

Cleavage pattern and survivin expression in porcine embryos by somatic cell nuclear transfer

Mammalian embryos produced in vitro show a high rate of early developmental failure. Numerous somatic cell nuclear transfer (SCNT) embryos undergo arrest and show abnormal gene expression in the early developmental stages. The purpose of this study was to analyze porcine SCNT embryo development and investigate the cause of porcine SCNT embryo arrest. The temporal cleavage pattern of porcine SCNT embryos was analyzed first, and the blastocyst origin at early developmental stage was identified. To investigate markers of arrest in the cleavage patterns of preimplantation SCNT embryos, the expression of survivin—the smallest member of the inhibitor of apoptosis (IAP) gene family, which suppresses apoptosis and regulates cell division—was compared between embryos showing normal cleavage and arrested embryos.A total of 511 SCNT embryos were used for cleavage pattern analysis. Twenty-four hours post activation (hpa), embryos were classified into five groups based on the cleavage stage as follows; 1-cell, 2-cell, 4-cell, 8-cell and fragmentation (frag). In addition, 48 hpa embryos were more strictly classified into 15 groups based on the cleavage stage of 24 hpa; 1-1 cell (24 hpa-48 hpa), 1-2 cell, 1-4 cell, 1-8 cell, 1 cell-frag, 2-2 cell, 2-4 cell, 2-8 cell, 2 cell-frag, 4-4 cell, 4-8 cell, 4 cell-frag, 8-8 cell, 8 cell-frag, and frag-frag. These groups were cultured until 7 d post activation, and were evaluated for blastocyst formation. At 24 hpa, the proportion of 2-cell stage was significantly higher (44.5%) than those in the other cleavage stages (1-cell: 13.4%; 4-cell: 17.9%; 8-cell: 10.3%; and frag: 13.9%). At 48 hpa, the proportion of embryos in the 2-4 cell stage was significantly higher (32.4%) than those in the other cleavage stages (2-8 cell: 8.2%; 4-8 cell: 12.1%; and frag-frag: 13.9%). Some embryos arrested at 48 hpa (1-1 cell: 5.8%; 2-2 cell: 2.8%; 4-4 cell: 3.8%; 8-8 cell: 6.5%; and total arrested embryos: 18.9%). Blastocyst formation rates were higher in 2-4 cell cleavage group (20.2%) than in other groups. SCNT embryos in 2-4 cell stage showed stable developmental competence. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-cell, 4-cell stage SCNT embryos were significantly higher than those of arrested embryos. Survivin protein expression showed a similar pattern to that of survivin mRNA. Normally cleaving embryos showed higher survivin protein expression levels than arrested embryos. These observations suggested that 2-4 cell cleaving embryos at 48 hpa have high developmental competence, and that embryonic arrest, which may be influenced by survivin expression in porcine SCNT embryos.     

Influences of retrieval stages and glutathione addition on post-thaw viability of quick frozen mouse morula during in vitro culture

Effects of the embryo retrieval stages and addition of glutathione (GSH) on post-thaw development of mouse morula were evaluated in 2 consecutive experiments. In the first experiment, 1-, 2-, 3- to 4- and 5- to 8-cell stage embryos were collected and cultured to the morula stage in Whitten's medium containing 0.1 mM ethylenediaminetetraacetic acid (EDTA). The development rate of 1-cell embryos to the morula stage was lower than that of the other stages (P<0.01). The post-thaw development rate of the morulae obtained from in vitro culture of 1-, 2-, 3- to 4-, and 5- to 8-cell embryos and from in vivo embryos (control) to the blastocyst stage was 55.5, 84.9, 87.4, 90.1 and 90.8%, respectively. The post-thaw development rate of morula obtained from in vitro produced 1-cell embryos was significantly lower than from the other stages or from the in vivo counterparts (P<0.0001). In Experiment 2, the impact of GSH supplementation of the culture medium in the presence or absence of EDTA was evaluated for embryo development to the morula stage and post-thaw survival, using in the 2 x 2 factorial design. Although EDTA supplementation increased development rates to the morulae (P<0.01) stage, GSH did not have an influence on morula development. However, the presence of either GSH or EDTA in the culture medium supported development to the blastocyst stage (P<0.01) of in vitro produced morulae. These data demonstrate that 1-cell embryos from a blocking-strain mouse cultured in vitro to the morula stage have a lower development rate following freezing and thawing than embryos collected at the 2-cell or later stages. Addition of EDTA or GSH, individually or in combination, to the culture medium may improve the development rate of morula to blastocyst stage following cryopreservation.