ASSESSMENT OF NUCLEAR TOTIPOTENCY OF FETAL BOVINE DIPLOID GERM-CELLS BY NUCLEAR TRANSFER

Nuclear transfer was used to study nuclear reprogramming of fetal dipl oid bovine germ cells collected at two stages of the fetal development . Ln the first case, germ cells of both sexes were collected during th eir period of intragonadal mitotic multiplication at 48 days post coit um (d.p.c.). In the second case, only male germ cells were collected a fter this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (24 7/511) cleaved; 2.7% (14/511) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed betwe en sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaugh tered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determine d, corresponded to that of donor fetus. Of 380 reconstituted embryos w ith male 105 to 185 d.p.c. germ cells, 72.1% (274/380) cleaved, 2.1% ( 8/380) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 whic h aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the a bsence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.