Effects of polyadenylation inhibition on meiosis progression in relation to the polyadenylation status of cyclins A2 and B1 during in vitro maturation of bovine oocytes

Abstract

The control of protein synthesis during maturation in oocytes is mainly exerted through cytoplasmic polyadenylation of stored mRNAs. We first analyzed the polyadenylation status of cyclins A2 and B1 during in vitro maturation (IVM) of bovine oocytes, using Rapid Amplification of cDNA Ends-Polyadenylation Technique (RACE-PAT). An inconstant elongation of the poly(A) tail was observed for cyclin A2 transcripts after maturation, while a constant lengthening was observed for cyclin B1, occurring during the first 12 hr of incubation. We then evaluated the effects of the polyadenylation inhibitor 3'-deoxyadenosine (3'-dA), on polyadenylation and nuclear maturation. The presence of 0.02 mM 3'-dA during the whole incubation period or from 6 hr after its beginning completely prevented meiosis progression in 100% of the oocytes. Polyadenylation of cyclin B1 was also completely prevented when 3'-dA was added at 0 hr, and greatly reduced when added at 6 hr. When 3'-dA was added at 12 hr, around metaphase I (MI), 46.9% of the oocytes have reached metaphase II (MII, vs. 78.8% in the control group) at 24 hr. The use of the same concentration of 3'-deoxyguanosine (3'-dG), that impairs transcription but not polyadenylation, did not affect cyclins polyadenylation, nor nuclear maturation, whatever was the timing of addition. These results suggest that the polyadenylation of cyclin B1 could be related to the first peak of activity of MPF, occurring around MI (10-12 hr after the onset of the maturation period). They also show that, in our culture conditions, inhibition of polyadenylation prevents meiosis progression, especially up to the MI stage, while inhibition of transcription does not.