Expression of 11 members of the BCL-2 family of apoptosis regulatory molecules during human preimplantation embryo development and fragmentation.
ABSTRACT Apoptosis during preimplantation development has received much interest because of its potential role in eliminating defective cells. Although development in humans is characterised by a high degree of genetic abnormality, little is known of the regulation of apoptosis in embryos. By PolyA PCR we analysed expression of 11 BCL-2 genes in individual human embryos representative of normal development and in severely fragmented embryos. We demonstrate constitutive expression of BAX in virtually all embryos at all stages of development, and variable expression of BCL2, BCL-XL, BCL-W, MCL-1 BAK, BAD, BOKL, BID, BIK, and BCL-XS. The frequency of expression of pro- and anti-apoptotic BCL-2 members was similar throughout development, except at the two-cell stage where pro-apoptotic genes predominated. Protein expression was confirmed for BCL-2, MCL-1, BCL-X, BAX, BAD, and activated caspase 3. BCL-2 protein was associated with mitochondria but expressed inconsistently in the blastocyst inner cell mass. Consistent differences between morphologically intact and fragmented embryos included the expression of BAK in fragmented but not intact four-cell embryos. Our study addresses the importance of examining single human embryos representative of the viable population for a large number of genes, in order to establish meaningful expression profiles and provide information on overlapping function in a large gene family.
Article: Interactions among activity of glucose-6-phosphate dehydrogenase in immature oocytes, expression of apoptosis-related genes Bcl-2 and Bax, and developmental competence following IVP in cattle.[show abstract] [hide abstract]
ABSTRACT: This study was conducted to understand whether the level of G6PDH activity assessed in immature bovine oocytes by means of BCB test was correlated with the level of expression of apoptosis-related genes such as Bcl-2 and Bax in immature and mature oocytes. This information should support previous findings suggesting that G6PDH activity is a useful marker for determining oocyte quality, thereby increasing the validity of BCB test in oocyte selection. Up to now, there are no data estimating the relation between G6PDH activity and the expression of apoptosis-related genes in oocytes. The expression of Bcl-2 and Bax genes was estimated on the mRNA and protein levels, respectively using real-time PCR and Western-blotting. To evaluate developmental competence of these oocytes, cumulus-oocyte complexes classified as BCB+ (low activity of G6PDH), BCB- (high activity of G6PDH) and Control were used for in vitro embryo production. In immature oocytes, the Bax transcript level in BCB- oocytes was significantly higher (P<0.001) in comparison to Control. In mature oocytes, the Bcl-2 transcript level was significantly lower in BCB+ oocytes (P<0.01) and in BCB- oocytes (P<0.05) in comparison to Control. However, no relation was found between the activity of G6PDH and the expression of the Bcl-2 or Bax proteins, both in immature and mature oocytes. Our results on the transcript level seem to indicate that oocytes subjected to BCB staining show tendency towards apoptosis. However, results obtained at the protein level did not confirm this conclusion. The usefulness of the BCB test as the indirect marker of apoptosis seems to be questionable. The lack of significant differences in the blastocyst rates developed from BCB+ and Control oocytes decreases the validity of BCB test in IVP technology.Theriogenology 03/2008; 69(5):546-55. · 1.96 Impact Factor
Article: Developmental changes in expression of genes involved in regulation of apoptosis in the bovine preimplantation embryo.[show abstract] [hide abstract]
ABSTRACT: The early bovine preimplantation embryo is resistant to proapoptotic signals until around the 8- to 16-cell stage. We hypothesized that 2-cell embryos have higher amounts of antiapoptotic proteins and lower amounts of proapoptotic proteins when compared to embryos ≥16 cells. Steady-state concentrations of mRNA for the antiapoptotic genes BCL2 and HSPA1A were higher for MII oocytes, 2-cell embryos, and 2-cell embryos treated with alpha-amanitin as compared to ≥16-cell embryos. Steady-state concentrations of mRNA for the proapoptotic gene BAD increased in embryos ≥16 cells. There was no significant effect of stage of development on steady-state mRNA concentrations of BCL2L1, DFFA, or BAX. Using immunohistochemistry, it was found that BCL2 was present in greater relative concentrations for 2-cell embryos than for embryos ≥16 cells. These results were confirmed by Western blotting. Relative amounts of immunoreactive BAX detected by immunofluorescence were lower for 2-cell embryos than for embryos ≥16 cells. Using Western blotting, a high molecular weight (46 kDa) form of BAX was highest in ≥16-cell embryos, intermediate in 2-cell embryos, and lowest in MII oocytes. There were no effects of stage of development on relative amounts of immunoreactive BCL2L1, HSPA1A, or BAD, as determined by immunofluorescence. Treatment of embryos with alpha-amanitin from Day 0 to Day 5 or Day 4 to Day 5 after insemination reduced activation of group II caspases and terminal deoxynucleotidyl transferase dUTP nick end labeling after treatment with the proapoptotic signal C(2) ceramide at Day 5 after fertilization. Thus, transcription of BAX or other proteins is required for acquisition of the capacity for apoptosis. Results support the idea that changes in amounts of BCL2 family members are important for the inhibition of apoptosis in the 2-cell embryo and in the establishment of the capacity for apoptosis later in development.Biology of Reproduction 01/2011; 84(1):43-51. · 4.01 Impact Factor
Article: Pathogenesis, developmental consequences, and clinical correlations of human embryo fragmentation.[show abstract] [hide abstract]
ABSTRACT: This narrative review summarizes the current state of knowledge about human embryo fragmentation during IVF. The clinical relevance of fragmentation is discussed and evidence supporting a central role for the oocyte in the pathogenesis of fragmentation is presented. A mechanism of fragmentation as aberrant cell division involving the cytoskeleton is described along with the novel concept of membrane instability in relation to follicular high-density lipoprotein metabolism and cholesterol transport.Fertility and sterility 12/2010; 95(4):1197-204. · 3.97 Impact Factor