112 THE PRESENCE OF LAMIN A/C ANTIGENS IN PORCINE EMBRYOS

Abstract

Nuclear lamins are components of the nuclear lamina, and their primary role is to support the nuclear envelope and provide anchorage sites for the chromatin. While type B lamins are expressed in all cells, type A lamins (including lamins A and C) are developmentally regulated and expressed in differentiated cells only. There are conflicting results about the presence of lamin A/C in early mammalian embryos. Lamin A/C was found to localize in the nuclear envelope of bovine, pig, and mouse embryos, while recently it has been reported that early mouse and bovine embryos lacked lamin A/C antigens. It has also been suggested that the existence of lamin A/C in the pronuclei of mouse and bovine nuclear transfer embryos indicated faulty reprogramming. The aim of this study was to investigate the presence of lamin A/C in porcine embryos of different origins (in vivo, parthenogenetic, and nuclear transfer). Embryos of various developmental stages were collected from inseminated gilts. For the production of parthenogenetic embryos, mature oocytes were electroporated and cultured for up to seven days. Fibroblast cells served as differentiated controls; progenitor cells from the olfactory bulb of a porcine fetus were used as undifferentiated controls. Lamin A/C was visualized by immunocytochemistry. Olfactory bulb progenitor cells lacked lamin A/C (0 out of 50 cells showed staining) while all fibroblast nuclei (n = 50) reacted positively with the antibody. GV-stage oocytes, being terminally differentiated cells, also possessed lamin A/C antigens (30/30). Lamin A/C was not detectable in any of the mature oocytes examined (n = 30), but it was found in early cleavage-stage embryos [both in vivo (20/20) and parthenogenetic (30/30)] with the signal becoming weaker in blastocysts (15/15). After nuclear transfer, the lamin A/C signal from fibroblast nuclei disappeared (20/20), consistent with nuclear envelope breakdown. Later it became detectable again; all nuclear transfer embryos reconstructed with either fibroblast or progenitor cells displayed lamin A/C staining in their pronuclei and at all stages examined (n = 65). This suggests that recipient oocytes remodel the donor nuclei and reassemble the nuclear envelopes of both differentiated and undifferentiated cells with type A lamins. Activated oocytes (n = 30) and early embryos (n = 40) were then incubated in the presence of actinomycin D (an inhibitor of RNA polymerase II) or cycloheximide (a protein synthesis inhibitor) for 14 h. Lamin A/C assembly was not perturbed by either treatment, indicating that the assembly did not result from de novo gene transcription but rather from solubilized lamins already in the cytoplasm. The results imply that lamin A/C is present in early pig embryos and that its presence after nuclear transfer is not an indicator of erroneous reprogramming, unlike that reported in cattle and mouse.