Porcine embryo development and fragmentation and their relation to apoptotic markers: a cinematographic and confocal laser scanning microscopic study

Abstract

Porcine embryo selection prior to transfer is mainly influenced by morphological criteria. However, the relationship between embryonic morphology, developmental potential and cell death by apoptosis in porcine embryos is still unclear. The aim of this study was to establish embryo quality parameters for in vivo fertilised porcine embryos based on timing of development in vitro, embryo morphology and the presence of apoptosis. The kinetics of development and morphological parameters were investigated in a time-lapse cinematographic experiment. Possible links between embryo morphology and apoptosis were examined via a confocal laser scanning experiment, analysing nuclear changes, annexin V and terminal dUTP nick-end labelling. The timing of early cleavages was firmly linked to embryo developmental competence in vitro. Attainment of at least the 5-cell stage before 77 h post insemination and attainment of the morula stage before 102 h post insemination significantly increased the odds for reaching the early blastocyst stage. Overall, a negative effect of fragmentation percentage and fragmentation pattern on subsequent embryonic development was observed, but the developmental potential of embryos experiencing slight fragmentation (0 -5%) was not different from embryos without fragmentation. Correlations detected between developmental arrest and fragmentation, and fragmentation and apoptosis were 0.60 and 0.87 (P < 0.05) respectively. Only a minority of the embryos arrested between the 1-and 4-cell stage displayed biochemical characteristics of apoptosis. Consequently, a significant correlation (0.57) between developmental arrest and apoptosis could only be established for embryos arrested after embryonic genome activation.

Temporal detection of caspase-3 and -7 in bovine in vitro produced embryos of different developmental capacity

“…The fluorescent detection of active caspases as early as the two-cell stage in our study has confirmed this possibility. Some of the two-cell embryos which stained positive for caspases and belonged to the slow developing group may have been in developmental arrest, which is in accordance with the previous studies where apoptosis has been demonstrated in arrested two-to four-cell human and pig embryos (Spanos et al 2002, Mateusen et al 2005. Notwithstanding the presence of the full apoptotic machinery in normal developing early stage embryos, in humans, caspase could only be detected from the compacted morula stage onwards (Spanos et al 2002).…”

“…Therefore, activation of caspases and fragmentation might not be correlated because apoptotic bodies are endpoints of apoptosis, whereas caspase activity occurs much earlier in the pathway. Thirdly, pattern and grade of fragmentation, which have not been evaluated in our study, may be responsible for different results, because minor fragmentation (below 15%) seems to have no effect on subsequent development in pigs (Mateusen et al 2005). Finally, the impact of asymmetry on embryo quality could be discussed, since it frequently disappears during in vitro pig embryo development (Mateusen et al 2005).…”

“…At each time point in the first experiment, symmetry and absence of fragmentation were used as morphological criteria, since both have been related to poor bovine embryo quality, lower pregnancy and implantation rates in cattle (Lindner & Wright 1983), and higher incidence of apoptosis in pigs (Mateusen et al 2005). Although abnormal morphology has been clearly associated with a higher apoptotic index and caspase expression in humans (Hardy et al 1989, Martinez et al 2002, and although fragmentation in humans can be associated with an expression of proapoptotic genes (Jurisicova et al 2003), abnormal morphology during embryo development could not be related to higher caspase activity in our study.…”

“…Superior embryo morphology is mainly characterized by evenly sized blastomeres and the absence of fragmentation (Lindner & Wright 1983) and is most frequently evaluated at the blastocyst stage (Bavister 1995). In human and porcine embryos, embryonic fragmentation is linked with apoptosis (Jurisicova et al 1996, Mateusen et al 2005; however, in vitro produced bovine morulae show no clear correlation between fragmentation and apoptosis, particularly in those embryos where fragments makeup !50% of the total embryo volume (Van Soom et al 2003). Further work is required to determine the underlying mechanisms surrounding embryonic fragmentation.…”

Effect of a porcine circovirus type 2 infection on embryos during early pregnancy

“…Another possible explanation for the fact that three embryos became negative for viral antigens 14 days after transfer may be related to apoptosis, the process that embryos use during mammalian preimplantation development to eliminate unwanted or damaged cells [28]. Since 48 h after the virus inoculation only a small proportion of the blastomeres (on average 6%) of the hatched blastocysts were infected [15], it is possible that through a confined apoptotical process this limited number of infected cells were expelled without any effect on further embryonic development [29]. Further research will focus on possible apoptotic effects PCV2 will have on embryos.…”