46 Presence of porcine TET3L isoform in oocytes: Potential involvement in the DNA demethylation process

Abstract

Fertilized oocytes undergo genome-wide DNA demethylation with the exception of imprinted genes and certain repetitive elements. Ten-eleven translocation 3 (TET3) protein has been known to be responsible for the DNA demethylation process by catalysing oxidation of 5-methylcytosine. Recent studies in the mouse indicated that multiple Tet3 isoforms exist in oocytes, implying differential actions of the isoforms. Previously, we reported the sequence of TET3 in porcine oocytes (GenBank: KC137685). Here, we investigated the presence of TET3 isoforms in porcine oocytes and cumulus cells, and followed changes in the TET3 expression during oocyte maturation to further understand the mechanism of TET3 action in the DNA demethylation process. To identify porcine TET3 isoforms, 5′ RACE (rapid amplification of cDNA ends) was conducted using mRNAs isolated from oocytes and cumulus cells at both germinal vesicle (GV) and MII stages. Gene-specific primers for 5′RACE were designed to recognise conserved regions of TET3 that are present in all isoforms, based on EST databases and the cloned sequence in our previous study. The PCR-amplified 5′RACE products were cloned into a vector and subsequently sequenced. The 5′RACE revealed 3 different TET3 isoforms from GV and MII cumulus cells; no amplification was detected using oocytes, potentially due to a low amount of mRNA. Among the isoforms, the longest variant (TET3L) contained sequences for the CXXC domain, known to carry DNA binding properties. Then, RT-PCR was used to detect the presence of the isoforms in porcine oocytes. Interestingly, the expression of TET3 isoform containing the CXXC domain (TET3L) was only verified from the RT-PCR, suggesting that the isoform may be the predominant isoform in porcine oocytes. To characterise transcript abundance of porcine TET3L, RNAs were isolated from different cells and/or tissues including cumulus cells, oocytes, brain, spleen, and lung. The RT-qPCR was performed using the RNAs and ^ΔΔCT method was used to analyse the data; GAPDH was used as an internal control. Three biological replicates were used for analysis of RT-qPCR data and P-values of less than 0.05 from one-way analysis of variance were considered significant. The expression level of TET3L was much higher in MII oocytes compared with that in somatic tissues; MII oocytes expressed the TET3 isoform over 350-fold higher than MII cumulus cells and 18-fold higher than lung cells. Interestingly, the expression level of TET3L increased over 3-fold during oocyte maturation (i.e. from GV to MII stage oocytes), indicating that TET3L may have a significant role in DNA demethylation after fertilisation. In conclusion, the TET3 isoform containing CXXC domain (TET3L) is predominantly expressed in matured porcine oocytes, suggesting an important role of the TET3 CXXC domain in DNA demethylation in zygotes.