110 NUCLEAR LAMIN A/C EXPRESSION IN BOVINE IVF EMBRYOS

Abstract

Lamin A/C expression is generally associated with terminally differentiated cell types; however, numerous conflicting reports in the literature demonstrate the presence of lamins A/C in pluripotent cells of pre-implantation embryos. This study characterized lamin A/C expression in bovine pre-implantation embryos using two monoclonal lamin A/C antibodies: anti-A/C IgM (A/C1) and anti-A/C IgG (A/C2) (Santa Cruz, California). Bovine embryos were produced as previously described (Fouladi-Nashta et al. 1998 Biol. Rep. 59, 255–262) and collected at various stages for immunofluorescence staining. Embryos were fixed in 100% methanol at −20°C for 20 min and then blocked for 1 h (4% goat serum in PBS) at RT. Samples were then incubated overnight at 4°C with mouse lamin A/C antibodies or with blocking solution as a control. Following the primary incubation, embryos were washed extensively in 1% BSA in PBS and then incubated with rabbit anti-mouse immunoglobulins (1:20) (DAKO, Denmark) for 1 h at RT. Unbound secondary antibody was removed by washing with 1% BSA in PBS, and embryos were counter-stained with 4',6-diamidino-2-phenylindole (2 μg/mL). Bovine fetal fibroblasts (BFF1) and human embryonic teratocarcinoma cells (EC1 and EC2) were processed identically to the embryos and used as positive and negative controls, respectively (Stewart and Burke 1987 Cell 51, 383–392). Images were viewed using epifluorescence (Leica DMR, Germany) and confocal microscopy (Leica TCS). BFF1 cells reacted with both lamin A/C1 and A/C2 antibodies. EC1 and EC2 stained positively for A/C2 whereas A/C1 was negative in both. All germinal vesicle (GV)-stage oocytes stained strongly for A/C2; however, for A/C1 only 67.5% were positive, and staining intensity was variable. Metaphase II oocytes stained negatively for both antibodies. One-cell zygotes exhibited a variable staining pattern similar to that of GV-stage oocytes. In contrast, all embryos from the 2-cell to blastocyst stage were negative for A/C1 but positive for A/C2. Our observations in embryos and EC cells indicate that the mouse anti-lamin A/C1 is specifically binding to lamin A/C whereas A/C2 is cross-reacting with other nuclear envelope proteins, possibly lamin B1/B2. The cross-reactivity of A/C2 has led to contradicting results in previous reports on lamin A/C expression in pre-implantation embryos. Our results with A/C1 show that lamin A/C is present in GV oocytes and 1-cell zygotes, suggesting that lamin A is important for pronuclear formation after fertilization. These results suggest that active remodelling of the nuclear envelope occurs during the early stages of bovine embryo development. RK is supported by a BBSRC postgraduate studentship. RA is a Marie-Curie Fellow.