Aurora Kinase A Is Not Involved in CPEB1 Phosphorylation and <i>cyclin B1</i> mRNA Polyadenylation during Meiotic Maturation of Porcine Oocytes

<div><p>Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither <i>cyclin B1</i> mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.</p></div

Detection of AURKA (Thr288) phosphorylation.

<p>(A) Fluorescent images of AURKA p-Thr288 localization detected by specific antibody in the GV- and MI-stage oocytes (28 h of IVM) and the oocytes cultivated for 28 h in the presence of 1 µM MLN8237. DNA is stained by DAPI. (B) A detail of chromosomes stained by AURKA p-Thr288 antibody in control MI-stage oocytes (28 h of IVM) and oocytes cultured for 28 h in medium supplemented with 1 µM MLN8237. Representative images from two independent experiments.</p

<i>Cyclin B1</i> mRNA polyadenylation and cyclin B1 expression after AURKA inhibition.

<p>(A) Polyadenylation of short (S-3′UTR) and long (L-3′UTR) forms of the <i>cyclin B1</i> mRNA was examined by poly(A)-test in oocytes collected before and after 28 h of IVM in media supplemented with stated concentrations of MLN8237. The polyadenylation is highlighted by white lines next to each lane. (B) Oocytes collected before IVM and after 28 h of IVM in media supplemented with stated concentrations of MLN8237 were subjected to western blot analysis of CPEB1, cyclin B1 and phospho-AURKA using specific antibodies. β-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as loading controls. The phosphorylated form of CPEB1 is marked (*). (C) The protein expression of cyclin B1 from six independent experiments was quantified using Quantity one software. The density of individual band was normalized to the total density of examined bands and to β-tubulin. The values represent the means ± SEM. No significant difference was detected between the control oocytes after 28 hours of IVM and the oocytes treated with different concentrations of MLN8237, P>0.05.</p

Morphological aspect of oocytes treated with MLN8237.

<p>Representative examples of morphological appearance of oocytes cultivated for 44 µM MLN8237. (A) Oocytes were fixed with ethanol:acetic acid (3:1) and stained by orcein. Sets of chromosomes are marked with arrowheads, a polar body is marked with and arrow. (B) Detail of chromosome configuration visualized by DAPI.</p

<i>In vitro</i> kinase assay – CPEB1 phosphorylation.

<p>(A) Oocytes were cultivated in control medium for 0, 28 or 44 h, Thereafter, <i>in vitro</i> kinase assay with CPEB1 peptide as an external substrate was performed in extracts prepared from 20 oocytes. MLN8237 was added to MI (28 h of IVM) oocyte extracts in final concentration of 10 µM in reaction buffer. Alternatively, recombinant AURKA (1 and 5 I.U. per assay) was used instead of oocyte extracts with or without MLN8237 in the concentration of 2 and 10 µM. The extracts were separated by SDS-PAGE and the phosphorylated substrates were visualized by autoradiography and analyzed using a BAS 2500 PhosphoImager. (B) <i>In vitro</i> kinase assay utilizing recombinant AURKA (1 I.U.) with or without MLN8237 in the concentration of 2 and 10 µM showing its activity towards either CPEB1 peptide as an external substrate, or LATS2 peptide used as a positive control. Phosphorylated substrates were separated by SDS-PAGE, visualized by autoradiography and analyzed using a BAS 2500 PhosphoImager.</p

Translation of luciferase reporter with <i>cyclin B1</i> 3′UTR in the presence or absence of MLN8237.

<p>(A) Schematic representation of porcine and murine <i>cyclin B1</i> 3′UTRs. ORF – open reading frame; CPE – cytoplasmic polyadenylation element; HEX – hexanucleotide, polyadenylation signal; PBE – Pumilio binding element. (B) A non-polyadenylated cRNA containing firefly luciferase open reading frame with murine <i>cyclin B1</i> 3′UTR was co-injected with polyadenylated <i>Renilla</i> luciferase cRNA as an internal injection control into GV-stage oocytes. Oocytes were cultivated for 24 and 28 hours in an inhibitor free medium (control) or the medium supplemented with 1 µM MLN8237. The firefly luciferase activity was normalized to the <i>Renilla</i> luciferase activity. The values are represented as means ± SEM. Data were subjected to t-test with Welch's correction. The differences between control and treated groups are not significant, P>0.05.</p