Utilization of cytoplasmic poly(A)+RNA for protein synthesis in preimplantation mouse embryos

The distribution of cytoplasmic poly(A)+RNA between subribosomal (less than 80S) and ribosomal/polysomal (greater than or equal to 80S) ribonucleoprotein particles has been investigated in mouse morulae and blastocysts. After labelling for 24h with [5,6-3H]uridine, late morulae (96h post-hCG), early blastocysts (100h post-hCG), or late blastocysts (120h post-hCG) were homogenized in detergent buffer, and 11 000g supernatants were prepared and centrifuged through 15-40% sucrose gradients. Poly(A)+RNA was isolated from the appropriate gradient fractions by affinity chromatography. In late morulae beginning to undergo cavitation, poly(A)+RNA was evenly distributed between the two types of RNP particles whereas it was almost entirely in the ribosomal/polysomal fraction in early and late blastocysts. The sedimentation profile (in 5-20% sucrose gradients) of poly(A)+RNA from the ribosomal/polysomal fraction of late morulae and blastocysts was the same, with a range of 4S to greater than 28S and a modal peak slightly smaller than 18S. Roughly 75% of this RNA was judged to be functional mRNA based on the EDTA sensitivity of the RNP particles containing it. Poly(A)+RNA from the subribosomal fraction of late morulae displayed a similar range of sedimentation values, but was enriched for a component sedimenting at 6-7S. These results demonstrate that the subcellular distribution of poly(A)+RNA shifts as cavitation begins, with the decline of the non-translating, subribosomal fraction. Although most of this fraction in late morulae is probably too small to constitute translatable mRNA, the remainder may represent a reserve available for recruitment into polyribosomes to support blastocyst expansion. Since little non-translating mRNA exists in blastocysts, the increasing rate of protein synthesis during blastocyst expansion must be driven by mRNA accumulation and/or stabilization.