In this report we demonstrate the successful in vitro culture of
fertilised embryos from 1-cell to blastocyst stage, albeit in a
strain-dependent fashion. We report procedures for the enucleation
of rat oocytes; nuclear transfer by injection of nuclei (NT) from
adult rat cumulus cells, rat primary embryonic fibroblasts and genetically
modified rat fibroblasts; and activation resulting in advanced
preimplantation development. Blastocyst stage rat embryos were obtained
after in vitro culture of nuclear transfer zygotes at similar frequencies
with each of these nuclear donor cell types. Transfer of NT embryos to
surrogate mothers leads to implantation of 24% of the zygotes. These
results suggest that the nuclei of cultured rat cells, even following
genetic modification, can be reprogrammed to support early embryonic
development, which is a prerequisite to cloning the rat.
The production of organ parenchyma in a rapid and reproducible manner is critical to normal development. In chimeras produced by the combination of genetically distinguishable tissues, mosaic patterns of cells derived from the combined genotypes can be visualized. These patterns comprise patches of contiguously similar genotypes and are different in different organs but similar in a given organ from individual to individual. Thus, the processes that produce the patterns are regulated and conserved. We have previously established that mosaic patches in multiple tissues are fractal, consistent with an iterative, recursive growth model with simple stereotypical division rules. Fractal dimensions of various tissues are consistent with algorithmic models in which changing a single variable (e.g. daughter cell placement after division) switches the mosaic pattern from islands to stripes of cells. Here we show that the spiral pattern previously observed in mouse cornea can also be visualized in rat chimeras. While it is generally held that the pattern is induced by stem cell division dynamics, there is an unexplained discrepancy in the speed of cellular migration and the emergence of the pattern. We demonstrate in chimeric rat corneas both island and striped patterns exist depending on the age of the animal. The patches that comprise the pattern are fractal, and the fractal dimension changes with the age of the animal and indicates the constraint in patch complexity as the spiral pattern emerges. The spiral patterns are consistent with a loxodrome. Such data are likely to be relevant to growth and cell division in organ systems and will help in understanding how organ parenchyma are generated and maintained from multipotent stem cell populations located in specific topographical locations within the organ. Ultimately, understanding algorithmic growth is likely to be essential in achieving organ regeneration in vivo or in vitro from stem cell populations.
A limiting factor in the development of new technologies and transport of rats worldwide has been the inability to robustly culture preimplantation embryos. Previously, culture in vitro to the blastocyst stage from one-cell embryos was successful only if the one-cell embryos were isolated near the time of the first cleavage and from only a few strains. Here we report the use of commonly available, chemically defined culture media to overcome these limitations. In vitro culture of young one-cell embryos using common embryo media (KSOM, BMOC, or HTF) for 18-22 h followed by culture in mR1ECM medium allows the successful in vitro development of blastocysts from one-cell embryos after 5 days from both outbred (SD) and inbred strains of rat (WF, LEW, F344, and PVG). This system allows the parthenogenetic development of chemically activated, unfertilized oocytes to the blastocyst stage. Embryos cultured in this system develop to term and are live-born following transfer to surrogate mothers.
The rat oocyte spontaneously activates under a wide variety of conditions. This process progresses to MIII arrest that is not responsive to parthenogenetic activation and development. Insofar as activation involves extrusion of the second polar body (PBII), we set out to determine if preventing this step by inhibiting microfilaments would change the course of spontaneous activation (SA). In particular, how long does the effect of SA persist while retaining reversibility of PBII extrusion once inhibitors are removed? We wanted to determine if the eggs would be responsive to parthenogenetic activation and capable of resuming development once a permanent inhibition is achieved. We set out to determine whether SA would depend on the ovular age of oocytes. Inhibiting of PBII extrusion was achieved by affecting microtubules with demecolcine or nocodazole or actin filaments with cytochalasin B (CB) and cytochalasin D (CD). We found that all oocytes undergo SA and progression to MIII; however, the rapidity of spontaneous activation is a function of the ovular age of the oocyte. The resumption of the meiosis period changes dramatically from 20 to 180 min with decreasing ovular age. We established that suppression of PB formation can be effectively achieved in oocytes of younger ovular age, and that inhibition of PB extrusion became irreversible after 3.5 h of treatment. We established that drug-treated oocytes could undergo subsequent reactivation and in vitro development to blastocysts. The rate of in vitro development of cytochalasin-treated group was comparable to parthenogenetic controls, while nocodazole and demecolcine produced oocytes that developed at lower frequencies. Thus, the application of the microfilament inhibiting drugs helps to overcome the negative effect of SA that results in MIII arrest. Here we also show optimized parthenogenetic stimulation that resulted in development to the blastocyst stage at frequency comparable to development of fertilized embryos.
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