The development of 1-cell mouse embryos in explanted oviducts, on mouse and bovine oviduct epithelial cells and on two established cell line supports is compared. The best rates of blastocyst formation were obtained using explanted oviducts; mouse and to a lesser extent, bovine oviduct epithelial cells allow good embryonic development, associated with high viability after transfer of the blastocysts obtained in co-culture. MDBK (from bovine kidney) and Vero (from Green monkey kidney) have been tested. MDBK allows high rates of blastocyst formation (67%) and the blastocysts obtained are viable. Vero does not allow the 2-cell block to be overcome. Maintenance of cell polarity for all the feeder layers did not improve embryo development. A preliminary study on the metabolic modifications induced by the feeder layers showed no modifications at all related to a decrease in glucose, an increase in lactate and early embryonic development. On the other hand, for the free amino acids, cellular supports with high embryotrophic activity seem to mimic tubal secretions, especially with a high level of glycine. Neither a genital tract origin, nor a hormonal contribution are strictly necessary for embryo co-culture, as already demonstrated by co-culture with trophoblastic tissue. Established cell lines, which are easy to handle and control, could be useful tools in embryo biotechnology.

This study proposes a procedure for the isolation and culture of oviduct epithelial cells of several species. In-vitro culture on such a feeder seems to allow full embryonic development and viability. The inner linings of Fallopian tubes from mouse, rabbit, cow and human were trypsinized and the epithelial cells were enriched with Percoll gradient. Isolated cells, obtained in high yield with good viability, were maintained in monolayer culture in B2-Menezo medium supplemented with serum, which also supports early embryonic development in vitro. The plated primary cultures reached confluence within 8 days, producing a monolayer of cohesive polygonal cells. Associated with this large epithelial cell population, ciliated cells as well as polykaryotic cells and few fibroblastic nests were observed. After the first sub-culture, the ciliated cells disappeared and the epithelial cell monolayer grew rapidly to confluence within 3 days and displayed contact inhibition. No epithelial cell growth could be obtained in culture in the absence of serum. The addition of oestrogens had no effect on any of the cultured oviductal epithelial cells. A spontaneous alteration was observed in morphology and growth after several passages, the number of which depends mainly upon the species.

In early 1997, the birth of a lamb after transfer of the nucleus from an adult mammary gland cell into an enucleated oocyte, along with the production of rhesus monkeys by nuclear transfer of embryonic cells, marked a reemergence of the field of mammalian cloning. Clonally derived rhesus monkeys would be invaluable in biomedical research, and the commercial interests in transgenic sheep and cattle propagated by cloning are substantial. Nuclear transfer technology is under consideration in human in vitro fertilization clinics to overcome infertility secondary to advanced maternal age or mitochondrial-based genetic disease. Nuclear transfer involves preparing a cytoplast as a recipient cell, in most cases a mature metaphase II oocyte from which the chromosomes have been removed. A donor nucleus cell is then placed between the zona and the cytoplast, and fusion, as well as cytoplast activation, is initiated by electrical stimulation. Successful reprogramming of the donor cell nucleus by the cytoplast is critical--a step that may be influenced by cell cycle stage. Embryos produced by nuclear transfer are cultured in vitro for several cell divisions before cryopreservation or transfer to the oviduct or uterus of a host mother. The efficiency of producing live young by nuclear transfer in domestic species is low, with a high frequency of developmental abnormalities in both preterm and term animals. However, a number of pregnancies have now been established using fetal cells as the source of donor nuclei. The use of cell lines not only allows large clone sizes but also supports the ability to genetically manipulate cells in vitro before nuclear transfer. Ongoing research focused on the production of clonally derived rhesus monkeys using fetal fibroblasts and embryonic stem cells as the source of donor nuclei will be reviewed.

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