Trophectoderm Nuclei Research Articles

A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes.

A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled. The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66). Differential labelling of TE and ICM nuclei in situ has two important advantages--that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.

The fate of inner cell mass and trophectoderm nuclei transplanted to fertilized mouse eggs.

When nuclei from 12–18-cell mouse embryos have been injected into fertilized mouse eggs1,2, donor and host chromosomes in those eggs that survived the operation combined to form a single tetraploid nucleus. Some of the resulting embryos developed into morphologically normal blas-tocysts. At the 8-cell stage, mouse blastomeres are still totipotent3 and are indistinguishable from one another. By the blastocyst stage, inner cell mass and trophectoderm have separated into distinct cell lineages that differ in morphology, physiology4 and biochemistry5. The fetus develops entirely from inner cell mass while the trophectoderm contributes only to placenta and fetal membranes; it is unclear when this is determined. Aggregation experiments suggest that some blastomeres are still labile at the late morula/early blastocyst stage6,7. If this was the case at least some of the donor cells used in my previous nuclear transfers might have been still totipotent. However I report here that nuclei from trophoblast and inner cell mass are different from one another, as judged by the developmental fate of fertilized mouse eggs to which they are transplanted.

Nuclear transplantation in mus musculus: Developmental potential of nuclei from preimplantation embryos

Genomic exchange of a mammalian cell has been attempted by entirely replacing the nuclear genome of the mouse egg with a nucleus from an embryonic cell via nuclear transplantation. Its biological applicability to experimental embryology has first been probed on the mouse blastocyst. After mechanical isolation of the trophectoderm (TE) and inner cell mass (ICM) from LT/Sv or CBA/H-T6 blastocysts and subsequent dissociation into single cells, their nuclei were transplanted singly into fertilized C57BL/6 eggs from which the female and male pronucleus were removed after nuclear injection. Of the 179 eggs injected with TE nuclei, 68 (38%) survived microsurgery and 34 cleaved, but most of them became arrested during early preimplantation development. Biochemical analysis of 25 TE nuclear-transplant embryos showed that nuclei from TE cells were not capable of initiating nor supporting development beyond preimplantation without participation of the egg genome. Of the 363 eggs injected with ICM nuclei, 142 (39%) survived micromanipulation and were cultured in vitro. From the 96 cleaving eggs, 48 embryos reached the late preimplantation stage. Biochemical or karyotypic analyses of 54 ICM nuclear-transplant embryos at various preimplantation stages for either strain-specific variants of glucosephosphate isomerase (GPI) or chromosomal markers (T6 translocation) revealed that, in most instances, the transplanted nuclei successfully promoted development of the recipient eggs. A total of 16 nuclear-transplant embryos originating from ICM nuclei were transferred into the uteri of pseudopregnant ICR/Swiss females in order to allow development to term. Three liveborn mice, two females and one male, were derived exclusively from the transplanted ICM nuclei as judged by their coat color, karyotype and GPI enzyme pattern. Furthermore, in subsequent matings, two of the nuclear-transplant mice, one female and one male, gave rise to several normal progeny all of which exhibited the nuclear-donor phenotype. Our results demonstrate that nuclei from ICM cells of the mouse blastocyst are not yet restricted in their differentiation capacities but rather remain developmentally equivalent to the totipotent zygote nucleus.