Chronological appearance of apoptosis in bovine embryos reconstructed by somatic cell nuclear transfer from quiescent granulosa cells.

Efficiency of cloning has remained low and in spite of attempts to improve this technology, many reconstructed embryos do not implant or are lost during early pregnancy. Chromosomal aberrations, deviant gene expression patterns and abnormal regulation of cell death may be involved in this increased early embryonic loss. Here, we investigate the chronological onset of both apoptotic changes in nuclear morphology and DNA degradation [detected by transferase-mediated dUTP nick-end labelling (TUNEL) reaction] in bovine two-cell- to blastocyst-stage embryos. Such embryos were generated either by reconstruction with nuclear transfer from quiescent granulosa cells or by regular in vitro embryo production. Nuclear condensation was observed from the two-cell stage and TUNEL labelling was observed from the six-cell stage in reconstructed embryos, whereas nuclear condensation was evident from the eight-cell stage and TUNEL labelling from the 13-cell stage in embryos derived in vitro. Furthermore, reconstructed embryos displayed elevated ratios of embryos containing apoptotic nuclei at pre-compaction stages and higher indices of apoptotic nuclei in morula and blastocyst stages when compared with in vitro-produced embryos.

Morphological assessment of preimplantation embryo quality in cattle.

The extensive use of embryo technologies has emphasized the need for assessing embryo quality by morphological techniques, such as transmission electron microscopy, immunocytochemistry for confocal laser scanning microscopy and fluorescence in situ hybridization. By a combination of these techniques, it has been possible to demonstrate: (i) that rRNA gene activation, as monitored by embryonic nucleolar development, is comparable in bovine embryos developed in vivo and produced in vitro, whereas reconstructed nuclear transfer embryos may be deviant, (ii) that generating embryos by both in vitro production and reconstruction by nuclear transfer is associated with increased occurrence of apoptosis, in particular in the inner cell mass of blastocysts, and (iii) that these two embryo production techniques are associated with increased occurrence of mixoploidy that is, embryos presenting a large population of normal diploid cells and a small population of abnormal haploid or polyploid cells. It is clear that blastocysts that appear healthy at stereomicroscopy may have subcellular defects. Therefore, the possibility of long-term evaluation in vitro of embryos after hatching has been examined. However, whereas embryos developing in vivo after hatching present a number of well defined developmental milestones, such as elongation of the trophoblast, formation of hypoblast and epiblast followed by differentiation of endoderm, mesoderm and ectoderm, in vitro culture systems for development beyond the blastocyst stage currently allow the embryo to complete only a single milestone, namely hypoblast formation.

Ultrastructure of oocyte maturation, fertilization, and early embryo development in vitro in the Siberian tiger (Panthera tigris altaica).

The application of assisted reproduction techniques to wild cats has been stalled by a lack of basic knowledge of the reproductive biology in these species. In this study, the ultrastructure of Siberian tiger (Panthera tigris altaica) cumulus-oocyte-complexes (COCs), as well as in vitro produced (IVP) zygotes and embryos were investigated, to estimate the normality of the manipulated reproduction processes. Adult female tigers were subjected to a purified porcine pFSH/pLH stimulation treatment followed by oocyte aspiration. According to morphological appearance at the stereomicroscopical level, COCs were classified as mature, immature, or degenerated, and then allocated into the following groups: presumptively immature COCs, which were in vitro matured (IVM-group) before fixation; presumptively mature COCs, which were either fixed after retrieval (pre-IVF-group), following in vitro insemination (IVF-group) or following in vitro insemination and subsequent in vitro culture (IVC-group). All specimens were processed for light and transmission electron microscopy (TEM). Both the IVM- and pre-IVF-group included oocytes in meiotic stages ranging from prophase I to metaphase II, and some prophase I oocytes in the IVM-group were apparently in their growth phase. The IVF-group presented features of presumptive normal fertilization, but aberrations such as polynucleation were also noted. The IVC-group included cleavage stage embryos of which, however, many were polynucleated. In conclusion, the procedures used for stimulation, aspiration, and classification of COCs resulted in retrieval of a heterogeneous population of oocytes which, following IVF and IVC, displayed a high rate of developmental deviations.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.