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Abstract
The mechanisms underlying the visual assessment and selection of immature oocytes
resulting in optimum embryonic development following in vitro maturation, fertilization
and culture (in vitro maturation (IVM)/in vitro fertilization (IVF)/in vitro embryo
culture (IVC)) are unknown. Also, the reasons for the more frequent occurrence of
cytoplasmic fragmentation in in vitro produced bovine embryos, resulting in poor survival
following cryopreservation and decreased pregnancy rates following embryo transfer
are not clear. The objectives of this study are: (1) to investigate whether differences
in the quality of immature oocytes and embryo fragmentation are associated with apoptosis;
and (2) to study the pattern of Bcl-2 and Bax expression in oocytes and embryos to
help elucidate their potential roles in the regulation of apoptosis during development.
Bovine oocytes were obtained from slaughterhouse ovaries and divided into four grades
(grades I-IV) based on their morphology. Oocytes of different grades were cultured
in serum-free medium for 48h. Embryos were produced only from grade I oocytes (highest
quality) via IVM, IVF and IVC procedures. The morphological analysis of apoptosis
in oocytes and embryos was carried out using propidium iodide staining and terminal
deoxynucleotidyl transferase mediated dUTP nick end labeling. The expression of Bcl-2
and Bax in oocytes and embryos of different qualities and stages was determined using
western blotting. The results showed that the number of morphologically abnormal oocytes
with shrinkage and/or fragmentation of the ooplasm, which are typical features of
apoptosis, was significantly higher in grade IV oocytes (denuded oocytes, the lowest
quality) than in grade I oocytes after 48h in vitro culture (P<0.05). DNA fragmentation,
a hallmark of the biochemical changes seen in apoptotic cell death, was observed in
morphologically fragmented oocytes and embryos. The expression of Bcl-2 was high in
good quality oocytes and embryos, low in fragmented embryos, and hardly detectable
in denuded oocytes. In contrast, the expression of Bax was found in all types of oocytes
and embryos with the highest expression in the denuded oocytes. This implies that
the ratio of Bcl-2 to Bax may be used to gauge the tendency of oocytes and embryos
towards either survival or apoptosis. Overall, our results show that apoptosis appears
to be an underlying mechanism of bovine oocyte degeneration and embryo fragmentation.
Interactions between the Bcl-2 family of proteins may play a critical role in pre-implantation
embryo development. These findings could have important implications for improving
IVF and related techniques.