Establishment of an embryonic stem cell line from 8-cell stage mouse embryos.

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.

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer.

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post coïtum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.

Nuclear transplantation using bovine primordial germ cells from male fetuses.

The developmental potential of nuclei of bovine gonial cells was investigated by nuclear transfer. Gonial cells were collected from male fetuses at about 175 days post coitum (p.c.). They were fused with enucleated oocytes; reconstituted embryos were cultured in vitro for 7 days. Embryos reaching the compacted morula or blastocyst stage were either fixed for cell counting or transferred into recipients. Out of 115 oocyte-gonia fusions, 101 (87.8%) gave rise to cleaved embryos at Day 3 and 26 (22.6%) had reached the 8-cell stage. At Day 7, 1 (1%) developed to the morula stage and 5 (4%) reached the blastocyst stage. Three blastocysts were fixed and showed normal cell numbers (135; 90; 76 cells). Three blastocysts and one morula were transferred in four recipients; two recipients were pregnant at Day 21 but only one was positive at Day 35 p.c.; this last one aborted around Day 40 p.c. No conceptus was collected. These results indicate that gonial cell nuclei can be partially reprogrammed; they are able to develop into blastocysts and to initiate gestation. However, more experiments will be necessary to prove the nuclear totipotency of bovine gonial cells.

Quantitative estimation of chimerism in mice using microsatellite markers.

An embryonic stem cell line was established from SV129 mouse blastocysts and used to generate chimeric mice by injection into OF1 blastocysts; 18 out of the 30 resulting offspring appeared chimeric as judged from their coat color patterns, and 3 of the 13 males proved to be germ-line chimeras as they transmitted the SV129 agouti phenotype to all or part of their offspring. The degree of chimerism of these males was evaluated for different tissues using polymorphic microsatellite markers amplified by the polymerase chain reaction. It was shown that these new markers can be effectively used to quantitatively estimate levels of chimerism. The CKMM (creatine kinase, muscle) microsatellite system was used to distinguish the SV129 from the OF1 genotype. In all performed tests, the correlation between DNA ratio and signal ratio, expressed as a base 10 logarithm, was shown to exceed or equal 0.98 for known DNA ratios (SV129/OF1) ranging from 1/99 to 99/1. Linear calibration methods were used to predict the % SV129 DNA of a test sample based on the obtained signal ratio. The accuracy of the prediction was evaluated by performing repeated measurements. Differences among three repeated estimates ranged from 2 to 17% for a given sample. Microsatellite systems should be very useful to monitor chimerism involving strains that can not be discerned with coat color or biochemical markers. This will be particularly important when ES methodology becomes available in species other than mice.

Developmental table of the early mouse post-implantation embryo.

The developmental tables of early somite mouse embryos that are presently available from the literature give clear and useful descriptions of the differentiation at successive stages. However, they provide no easy access to the correlation between the growth of the embryo and its differentiation. In the present study, quantitative data concerning normal mouse embryonic development as well as the major developmental events occurring between 0 and 30 pairs of somites were established. Measurements of growth (crown-rump length, head length, absorbancy at 280 nm) and differentiation parameters (morphological score) of 168 to 310 explanted mouse embryos were recorded for each developmental stage (number of pairs of somites). A short description of the major events occurring at the corresponding stages is also presented. The table is more detailed than those presently available and provides a rapid and practical overview of the timing of the appearance of developmental events and differentiation in correlation to the progressive growth of the embryo. It could, therefore, be useful for embryologists and toxicologists. In addition, the development of 67 post-implantation mouse embryos cultured in vitro was compared with the reference table established from in vivo embryos. Our results confirm and extend previous reports showing that embryos cultured in vitro grow and differentiate at a pace very similar to that of embryos developed in vivo.

Evolution of the developmental scores of sixteen morphological features in mouse embryos displaying 0 to 30 somites.

A precise framework of morphological developmental events observed macroscopically in early postimplantation mouse embryos aged 8-10 days (0-30 somites) is established. The quantitative evolution of the developmental score of 16 features as a function of the developmental stage of the embryos (expressed in number of somites) is presented. Thirty-one groups of ten embryos, each with 0 to 30 somites, were scored for each feature according to the previous description of the authors. In addition, the variation of individual structures as a function of embryonic developmental stages is evaluated. It is suggested that the framework of differentiating individual structures at given developmental stages will help to plan experiments in developmental biology of rodents and will facilitate the interpretation of results in developmental toxicity.

Interlaboratory evaluation of three culture media for postimplantation rodent embryos.

The first aim of the study was to compare the ability of rat serum, human serum, and a mixture of human and rat serum (4:1) to support in vitro development of rodent postimplantation embryos. The comparison was made in three laboratories using rat embryos and in one laboratory using mouse embryos. Batches of sera, initial developmental stage, duration of culture, and endpoints were identical in the laboratories. The second aim of the study was to evaluate if other variables that could not be standardized would significantly influence the results of the laboratories. No reproducible difference was observed among the culture media or among the laboratories except that growth and differentiation were slower in the laboratory using mouse embryos. Further experiments are needed to exclude small differences in performance of the media.

Morphogenesis and quantification of the development of post-implantation mouse embryos.

This paper describes potential improvements in the quantitative assessment of the differentiation of rodent embryos used for in vitro embryotoxicity studies. A chart of schematic illustrations of the developmental stages of seventeen morphological features observed macroscopically in mouse embryos aged 8-10 days (0-30 somites) has been drawn up. The chart is based on the morphological scoring system proposed by Brown and Fabro (1981) for rat embryos and complements the original descriptions. Some intermediate stages have been added to the scoring system. The original and the modified scoring systems were applied to 310 mouse embryos in 31 groups of ten embryos, each with 0 to 30 somites. The modified score is consistently about 25% higher than the original score. The correlation of both the original and the modified scores with the number of somites is best expressed by an asymmetric sigmoid. The chart and the modified scoring system could also be used, with minor adaptations, to assess rat embryos at corresponding developmental stages.