Involvement of polyadenylation status on maternal gene expression during in vitro maturation of porcine oocytes.
During mammalian oocyte maturation, protein synthesis is mainly controlled through cytoplasmic polyadenylation of stored maternal mRNAs. In this study, the role of polyadenylation modification of maternal transcripts in pig oocytes was investigated by adding cordycepin (3'-dA), a potent polyadenylation inhibitor, to the culture medium of porcine oocytes maturing in vitro. 3'-dA significantly prevented cumulus expansion regardless of the concentration used, and inhibited pig oocyte maturation in a dose-dependent manner. Further, 3'-dA 1 microg/ml-treated MII oocytes experienced significantly lower rates of cleavage (29%) and blastocyst formation (15.35%) compared to control MII oocytes (58.6% and 35.3%, respectively). Western blotting revealed that the activity of mitogen-activated protein kinase (MAPK) and p34(cdc2) was significantly decreased in oocytes and cumulus cells treated with 3'-dA at a concentration of 1 microg/ml or greater. To further explore the underlying molecular mechanisms, expression patterns and polyadenylation states of four important genes, C-mos, cyclin B, GDF9 and BMP15, were studied as representative maternal transcripts by real-time PCR and the PAT assay. 3'-dA at concentrations above 1 microg/ml significantly prevented polyadenylation and caused aberrant expression of C-mos and GDF9 during oocyte maturation. These results suggest that polyadenylation inhibitor blocked pig oocyte maturation in vitro by one or more of the following actions: (1) inactivation of MAPK and MPF in oocytes, especially at the late stages (MI and MII); (2) prevention of cumulus cell expansion through inactivation of cellular MAPK; and (3) inhibition of the maternal mRNA polyadenylation process, which in reverse, disrupted the maternal mRNA patterns in pig oocytes' maturation in vitro.
Zhang
Ding-Xiao
DX
Department of Animal Sciences, Chungbuk National University, Cheongju, Chungbuk 361-763 Korea, Cheongju, 361-763 South Korea.
Cui
Xiang-Shun
XS
Kim
Nam-Hyung
NH
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D054885:Cumulus Cells / Q000187:drug effects; D003839:Deoxyadenosines / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D009153:Mutagens / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D011061:Poly A / Q000378:metabolism; D026723:Polyadenylation; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine
D003839:Deoxyadenosines; D009153:Mutagens; D012333:RNA, Messenger; D011061:Poly A; D016203:CDC2 Protein Kinase; D048049:Extracellular Signal-Regulated MAP Kinases; C058120:cordycepin