45 NUCLEAR LAMIN A/C EXPRESSION IN BOVINE PARTHENOTES AND NUCLEAR TRANSFER EMBRYOS

Abstract

Despite the apparent successes of nuclear transfer (NT) technology, numerous recent reports have indicated de-regulation of key gene expression patterns in NT embryos as compared to their in vivo and IVF counterparts. Aberrant expression of lamin A/C has been reported in mouse (Moreira et al. 2003 J. Cell Sci. 116, 3713-3720) and bovine (Sullivan et al. 2004 Biol. Rep. 70, 146-153) NT embryos, leading to the hypothesis that the presence of lamin A/C might affect subsequent development. Lamin A/C expression is a potential marker for reprogramming due to the induced expression and remodeling during differentiation. Previously using immunofluorescence in bovine IVF embryos, we have demonstrated the persistence of lamin A/C until the 2-cell stage (Kelly et al. 2005 Reprod. Fertil. Dev. 17, 205-206). The present study was initiated to further characterize lamin A/C expression in bovine parthenogenetic and NT embryos using a monoclonal antibody specific to lamin A/C. Bovine oocytes were matured in vitro as previously described (Fouladi-Nashta et al. 1998 Biol. Rep. 59, 255-262). NT embryos were constructed using lamin A/C-positive primary bovine fetal fibroblasts and in vitro-matured, enucleated MII bovine oocytes. Oocyte cell couplets were fused at 24 h post onset of maturation 1 h prior to activation. Oocyte activation was achieved with 7% ethanol for 7 min followed by a 6 h incubation in mSOF containing 10 µg/mL cycloheximide and 7.5 µg/mL cytochalasin B for the production of both NT and parthenogenetic embryos. Embryos were cultured in mSOF supplemented with 10% FCS and collected at various stages for immunofluorescence staining. Prior to fixation, embryos were incubated in 2 mg/mL protease to remove the zona pellucida. Samples were fixed in 100% methanol at -20°C for 20 min and then blocked for 1 h (4% goat serum in PBS) at RT. Embryos were then incubated overnight at 4°C with mouse anti-lamin A/C antibody (IgM; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with blocking solution as a control. Following the primary incubation, embryos were washed extensively in 1% BSA in PBS and then incubated with Cy3 goat anti-mouse IgM (1:400) (Chemicon International, Inc., Temecula, CA, USA) for 1 h at RT. Unbound secondary antibody was removed by washing with 1% BSA in PBS, and embryos were mounted in VectaShield mounting medium containing 42,6-diamidino-1-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were viewed using epifluorescence (Leica DMR; Leica Microsystems, Wetzlar, Germany) and confocal microscopy (Leica TCS). Inhibiting protein synthesis during the activation period with cycloheximide had no effect on lamin A/C assembly in 6 h post activation (hpa) parthenogenetic (35/35) and NT (7/7) embryos. The pronuclei of parthenogenetic (30/30) and NT (15/15) zygotes at 22 hpa were also positively labeled for lamin A/C. Nuclear labeling was observed in both parthenogenetic (25/25) and NT (12/12) 2-cell embryos. All parthenogenetic and NT embryos examined from the 4-cell stage through to blastocysts were stained negatively for lamin A/C. These results suggest that lamin A/C present in bovine NT zygotes is not due to aberrant reprogramming and that remodeling of the nuclear lamina occurs correctly in bovine NT embryos.