Characterization of calcium oscillation patterns in caprine oocytes induced by IVF or an activation technique used in nuclear transfer.

Routine activation of nuclear transfer (NT) eggs involves the application of a single intracellular calcium [Ca2+]i rise, stimulated by an electrical pulse, as opposed to [Ca2+]i oscillations, which is the natural mode of sperm-induced activation at fertilization in all mammalian species tested to date. It has yet to be shown that caprine oocytes exhibit an increase in calcium at fertilization in a manner similar to other mammals. The objective of the present study was to evaluate and characterize the ([Ca2+]i) oscillation patterns of caprine metaphase II (MII) oocytes during IVF and during an activation techniques used in nuclear transfer. Additionally, the effect of cytochalasin B (cyto B) in the NT process was evaluated for its impact on [Ca2+]i oscillations and subsequent embryo development. Mature in vitro and in vivo derived caprine oocytes were activated by 5 microM ionomycin, an electrical pulse(s), or IVF. The intracellular Ca2+ response was determined using the [Ca2+]i indicator Fura-2 dextran (Fura-2D). Ova treated with ionomycin or stimulated by an electrical pulse exhibited a single [Ca2+]i rise, whereas IVF-derived oocytes showed oscillations. IVF [Ca2+]i showed some variation, with 62% of in vitro matured oocytes exhibiting oscillations, whereas 8% of in vivo matured oocytes exhibited oscillations demonstrating a correlation between [Ca2+]i responses and maturation technique. Knowing the [Ca2+]i profile of activated eggs, one may be able to optimize the activation methodology used in a production nuclear transfer setting which could potentially improve development to term for NT embryos.

Effect of serum concentration, method of trypsinization and fusion/activation utilizing transfected fetal cells to generate transgenic dairy goats by somatic cell nuclear transfer.

This work was performed within a commercial nuclear transfer program to investigate different methods for synchronizing donor cell cycle stage, for harvesting donor cells, and for fusion and activation of reconstructed caprine embryos. Primary fetal cells isolated from day 35 to day 40 fetuses were co-transfected with DNA fragments encoding both the heavy and light immunoglobulin chains of three different monoclonal antibodies and neomycin resistance. Four neomycin resistant cell lines for each antibody were selected, expanded, and aliquots were both cryopreserved for later use as karyoplast donors or used for further genetic characterization. Transfected fetal cells were cultured in 0.5% FBS to synchronize G0/G1 cell cycle stage cells, then re-fed with 10% FBS prior to use to allow donor cells to re-enter the cell cycle. Alternatively, transfected fetal cells were grown to confluence in 10% FBS to induce contact inhibition to synchronize G0/G1 cell cycle stage cells. Adherent monolayers of transfected fetal donor cells were harvested by either partial or complete trypsinization. Donor cells were simultaneously fused and activated with enulceated in vivo produced ovulated oocytes from superovulated does. Half of the fused couplets received an additional electrical activation pulse and non-fused couplets were re-fused. Four live offspring were produced from 587 embryos generated from cell lines cultured in 0.5% FBS, while one live offspring was produced from 315 embryos generated from cell lines cultured in 10% FBS (0.7% versus 0.3% embryos transferred, respectively, P > 0.05). Five offspring were produced from 633 embryos generated from cell lines harvested by partial trypsinization (0.8% embryos transferred), and no offspring were produced from 269 embryos generated from cell lines harvested by complete trypsinization. Four live offspring were produced from 447 embryos generated from re-fused couplets, and one live offspring was produced from 230 embryos generated from fused couplets that received an additional electrical activation pulse (0.9% versus 0.4% embryos transferred, respectively, P > 0.05). These results suggest that low-serum culture of transfected goat fetal cells and harvest by partial trypsinization may be more efficient methods for generating transgenic goats by somatic cell nuclear transfer. In addition, re-fusion of non-fused couplet or an additional activation step was successful for producing live offspring.

The use of fluorescein isothiocyanate (FITC) as a short-term cell lineage marker in the peri-implantation mouse embryo.

Fluorescein isothiocyanate (FITC) may prove to be a useful short-term cell lineage marker in the early mouse embryo. Blastomeres and embryos are labelled by a 10 min exposure to 0.5 mg/ml FITC in ungassed medium 16 containing 2 mg/ml polyvinylpyrrolidone. FITC-labelled embryos divide at rates comparable with control non-labelled embryos, undergo polarization and cell flattening at compaction at the 8-cell stage, generate distinct inner and outer cell populations at the 16-cell stage and form blastocysts with both ICM and trophectodermal tissues. The label is equally transmitted to all progeny of a labelled cell, is stable in the cells for several days and is not transferred to neighboring non-labelled cells via gap junctions. The fluorescent labelling observed is predominantly cytoplasmic and may reflect an unusual permeability of embryonic plasma membranes.

Properties of polar and apolar cells from the 16-cell mouse morula.

The surface properties of newly formed, isolated 1/16 mouse blastomeres have been analyzed over the 10-12 h period prior to their division to 2/32 cells. Two populations of cells are formed at the 8- to 16-cell transition and their surface phenotypes vary with their relative position within the morula. Outer cells are polar, relatively non-adhesive and relatively large; inner cells are apolar, adhesive and smaller. The surface phenotypes of both inner and outer 1/16 cells are stable during culture for 11 h in isolation. However, the surface phenotypes can be induced to change by culture in combination with a second 1/16 cell, in a manner that is dependent upon the identity of the second cell. Two aggregated polar cells never flatten completely against each other, and both cells retain a clearly defined polar phenotype for 11-12 h. In aggregates of two apolar cells, cell outlines are lost as a result of intercellular flattening and microvilli are displaced away from areas of cell contact. However, if the two apolar cells are subsequently separated an even distribution of microvilli is restored. In most aggregates of an apolar and a polar cell, the polar cell envelops the apolar cell completely. These results are discussed in the context of the normal fate and potential of each cell type within the morula.