52 NUCLEAR REMODELING AFTER SOMATIC CELL NUCLEAR TRANSFER (SCNT) IN THE RHESUS MONKEY

Abstract

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. MPF degradation, caused by fertilization or artificial activation, is in turn required for pronuclear formation and subsequent embryonic cleavage, and involves cyclin B catalyzed proteolysis by the proteasome system. In the rhesus monkey, SCNT results in the production of cleaving embryos, but development arrests at the morula stage presumably because of abnormal or incomplete reprogramming. We undertook this pilot study to examine the timing and extent of nuclear remodeling events (NEBD, PCC) in monkey SCNT embryos. The proteasome inhibitor MG-132 was employed to avoid or delay premature MPF degradation (Zhou et al. 2003 Science 302, 1179). Monkey fetal fibroblasts employed as nuclear donor cells were fused with nonactivated cytoplasts and incubated in the presence (n = 20) or absence (control; n = 35) of MG-132. Embryos were fixed and co-labeled with DAPI (DNA) and monoclonal antibody against lamin A/C (nuclear envelope). In monkey germinal vesicle-stage oocytes (n = 5) and zygotes (n = 6), a lamin A/C signal was detected at the nuclear periphery while matured MI (n = 6) and MII (n = 12) oocytes were negative for lamin A/C staining, consistent with the absence of a nuclear membrane. Donor fetal fibroblasts arrested at the G1 stage of the cell cycle exhibited a lamin A/C signal. Minimal or no changes were observed in donor nuclei within 1 h after fusion. The majority of control SCNT embryos sampled 4 h after fusion exhibited only slight chromatin condensation; however, they failed to form metaphase chromosomes. Positive lamin A/C staining indicated the presence of intact nuclear membranes. Following activation these SCNT embryos cleaved, but arrested at the 8–16 cell stage. In initial experimentation we determined the minimal efficient concentration of MG-132 to be 5 μM, that is capable of inhibiting first polar body extrusion during the MI–MII transition. Subsequent in vitro development to the blastocyst stage (53%) of fertilized oocytes treated with 5 μM MG-132 for up to 4 h was similar to that of nontreated controls. Incubation of SCNT embryos (n = 20) for 4 h with MG-132 resulted in robust chromosome condensation, spindle formation, and weak or partial lamin A/C signal. Our observations suggest that incomplete nuclear remodeling events in monkey SCNT embryos may be due to premature MPF inactivation perhaps caused by the fusion pulse. Future studies will address the developmental potential of monkey SCNT embryos exposed to MG-132.

This work was supported by NIH grant NS04330, Core Grant RR00163 and a product donation from Ares Advanced Technology, Inc.