Laminin chain-specific gene expression during mouse oocyte maturation.
Expression patterns of laminin chain mRNAs (A, B1, and B2) during mouse oocyte maturation were examined using the competitive reverse transcription-polymerase chain reaction (RT-PCR) method. Total and poly (A)-rich mRNAs isolated from various stages of maturing oocytes in vitro were subjected to RT-PCR and the precise amount of laminin chain-specific mRNA transcripts was estimated by adding externally known amounts of in vitro transcribed mutant cRNA transcripts as an internal control. The estimated copy numbers for A, B1, and B2 chain mRNAs in a single germinal vesicle-stage oocyte were 1.34 +/- 0.19 x 10(5), 6.95 +/- 0.32 x 10(6), and 2.0 +/- 0.56 x 10(5), respectively. Although notable changes of all laminin chain mRNA levels were not observed at any stage of meiotic maturation in total mRNA preparation, chain- and meiotic stage-dependent alterations of poly (A)-tailed mRNA quantities were observed in poly (A)-rich mRNA preparation. A potent RNA synthesis blocker, alpha-amanitin did not influence the changes of mRNA levels, implying the presence of posttranscriptional regulation mechanism in the expression of laminin chains during mouse oocyte maturation. Discrete and time-dependent deadenylation of A and B1 chain, but not B2 chain mRNA, was observed during oocyte maturation by a rapid amplification of cDNA ends (RACE)-PCR. In germinal vesicle (GV)-stage oocytes, only B1 chain was found to be present in a highly polyadenylated state and subsequent deadenylation was observed as meiosis progressed. The poly (A) tail modification was dependent on the initiation of meiotic resumption. Although all laminin chain mRNAs were found in fully grown and meiotically competent mouse oocytes, Western blot analysis detected the B1 chain polypeptide only in GV- and polar body (PB)-stage eggs. These results suggest that the expression of laminin B1 chain in mouse oocytes may be due to its large amount of mRNA transcripts and/or high level of polyadenylation state that is efficient for translational activation.
Shim
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C
Department of Molecular Biology, College of Natural Sciences, Seoul National University, Korea.
Lee
S G
SG
Song
W K
WK
Lee
C S
CS
Lee
K K
KK
Kim
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K
D000818:Animals; D015153:Blotting, Western; D015870:Gene Expression; D007797:Laminin / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D009865:Oocytes / Q000254:growth & development / Q000378:metabolism*; D009866:Oogenesis; D016133:Polymerase Chain Reaction; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism
D007797:Laminin; D012333:RNA, Messenger