72 LIMITED REPROGRAMMING OF SOMATIC CELL NUCLEI TRANSFERRED INTO MOUSE IMMATURE OOCYTES

Abstract

Somatic cell nuclear transfer, an important approach for the analysis of certain functional changes in the genome during differentiation and for many practical applications, is in general a low-efficiency procedure, mainly due to a low effectivity in the re-establishment of the developmental program in the reconstructed embryo. The process of reprogramming is, however, poorly understood and some additional studies are clearly necessary. The aim of this study was the ultrastructural and immunofluorescent (B23-nucleophosmin) evaluation of somatic (cumulus) cell nuclei reprogramming after their transfer into intact immature mouse oocytes, kept at the germinal vesicle (GV) stage (dbcAMP) during the whole culture. Control somatic cells and nuclear transfer-reconstructed embryos (1 and 24 h after fusion induced by polyethyleneglycol) were fixed for transmission electron mcroscopy (TEM) in 2.5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated through an ethanol series, and embedded in epoxy resin. Finally, ultrathin sections were stained with uranyl acetate followed by lead citrate. The above reagents were purchased from Electron Microscopy Sciences (Hatfield, PA, USA). In parallel, we have evaluated immunocytochemically the pattern of B23 labelling in intact and reconstructed cells. The samples were fixed in 4% paraformaldehyde, incubated with an antibody against B23 (Santa Cruz, CA, USA) and then with a biotinylated secondary antibody, and detected by fluorescein isothiocyanate (FITC)-coupled Streptavidin (Jackson ImmunoResearch, Cambridgeshire, UK). Mouse cumulus cells (12/12) contain a reticulated fibrillogranular nucleolus. The cell are also positively labeled with the anti-B23 antibody. One hour after fusion, the introduced nuclei displayed shape modifications and nuclear envelope irregularities, whereas the nucleolus still showed the typical fibrillar pattern (19/19). The volume of transferred nuclei remains unchanged. Interestingly, while the oocyte nucleolus remains negative for B23, the nucleoli in transferred somatic cells were always positively labeled (45 cells). After 24 h, the transferred nuclei increased their volume up to two-three times and displayed an irregular shape with nucleoli still possessing the unchanged reticulated pattern (17/17). As in the previous experimental interval, only the somatic cell nucleus remained labeled with the anti-B23 antibody (52 cells). The GV oocyte nucleoli remained unchanged during the whole culture period, exhibiting the typical dense-fibrillar pattern. Our results showed that the immature oocyte cytoplasm possesses a limited remodelling activity. Interestingly, the evident increase in volume of transferred somatic cells indicated some changes but TEM morphology and B23 labeling pattern remained basically unchanged. We cannot, however, exclude the beneficial effect upon reprogramming if these nuclei were subsequently used for the transfer into definitive cytoplasts.

This work was supported by ESF STE/05/E004.