Characterization of deoxyribonucleic acid synthesis and the transition from maternal to embryonic control in the 4-cell porcine embryo

Abstract

These studies were conducted to identify the point during the 4-cell stage at which the porcine embryo begins to control development. Reproductive tracts of gilts were flushed 48 h after the onset of estrus to obtain 1- and 2-cell embryos. To determine the duration of the 4-cell stage in vitro, development of 29 embryos was timed from cleavage to the 4-cell stage and from cleavage to the 8-cell stage. The average duration of the 4-cell stage was 50.5 h. The duration of the 4-cell stage was positively correlated (p < 0.01) with culture time in vitro before cleavage to the 4-cell stage. DNA content was determined by using the Feulgen's reaction and quantified with micro-densitometry. Staining units (SU; density x area) were calculated at 0, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, and 36 h post-cleavage to the 4-cell stage (P4C). Results revealed a possible G1 phase (< 2 h) with DNA synthesis starting within 2 h P4C. DNA synthesis was completed by 16 h P4C, and was followed by an extended G2 phase. Embryos were evaluated for uptake and incorporation of [35S]methionine and for qualitative changes in protein profiles specific to time points during the 4-cell stage (2, 10, 14, 16, 18, 24, 30, and 40 h P4C). Methionine uptake and incorporation into protein followed similar patterns, both decreasing until 16-18 h P4C, followed by a steady increase through the 4-cell stage. Protein profiles revealed qualitative changes beginning at 14 and 16 h P4C. Four-cell stage embryos from various time points P4C (0, 4, 8, 12, 16, 20, and 24 h P4C) were cultured in α-amanitin until 24 h P4C, when all embryos were radiolabeled. Uptake and incorporation of [35S]methionine followed similar patterns, with both decreasing until 16 h P4C, followed by a steady increase through the 4- cell stage. A qualitative change began to occur when α-amanitin was added at 12 h P4C, with most qualitative changes complete by 16 h P4C. Embryos completed the third cell cycle when continuously cultured in the presence of α-amanitin from 24, 30, and 36 h P4C. These results suggest that the maternal genome controls development until 16 h P4C (after completion of DNA synthesis) and that the embryonic genome begins to control development at the start of the G2 phase.