Study On The Expression Of SMYD3 Gene And Its Function During Bovine Oocyte Maturation In Vitro

SMYD3(SET and MYND domain-containing protein 3) is a histone H3K4-specific dimethyltransferase and trimethyltransferase, having two conserved SET and MYND domains, the SET domain acts as methyltransferase and the MYND domain is a zinc finger motif which is involved in protein-protein interaction and recognizes specific sequence of DNA. SMYD3 can regulate the activity and expression of downstream genes by binding their promoter and maintaining H3K4 trimethylation which is associated with active transcription, including telomerase reverse transcriptase (TERT) gene and so on. Only two H3K4-specific trimethyltransferases were found recently, which are SMYD3 and Meisetz. H3K4 was found trimethyled through bovine oocyte maturation. The transcript of Meisetz was detected only in germ cells when meiosis proceeded from pre-meiotic stage to pachytene and not found after pachytene. It is implied that there are other methyltransferases maintaining H3K4me3 modification status, and SMYD3 is potential candidate.The regulatory mechanism of SMYD3 in cancer cells is investigated extensively, while lack of knowledge in bovine oocyte maturation, fertilization and embryo development. This study detected the dynamic changes of SMYD3 mRNA and protein during bovine oocyte maturation. The RNAi was used to investigate the effects of knockdown of SMYD3 mRNA on the bovine oocyte maturation, fertilization, and embryo development by microinjecting three SMYD3-siRNAs into GV bovine oocytes and zygotes.1. Dynamic changes of the SMYD3 mRNA and protein during bovine oocyte in vitro maturation(IVM)In our culture system, there were 82.9%, 80.3%, 75.1%, 63.3%, 70.9% of ooctyes having reached GV,PMI,MI,AI-TI and MII respectively at 0,8,12,18,24 hr after culture in vitro. As a result, we collected GV,PMI,MI,AI-TI and MII ooctyes according to the culture time in vitro.Real-Time PCR results showed that SMYD3 was transcripted during bovine oocytes IVM. Compared with GV oocytes, the relative amount of SMYD3 mRNA remarkably elevated(P<0.05)from MI to AI-TI and then slowly decreased from AI-TI to MII (P>0.05). The immunochemistry results showed the protein of SMYD3 began to be translated at AI-TI, and gradually increased from AI-TI to MII, and distributed with chromosomes. It is implied that the accumulation of SMYD3 mRNA finished before AI-TI, and the protein is translated and begin to be active at AI-TI. SMYD3 is presumed to play a key role in maintaining of H3K4me3 after AI-TI.In this study, an unreported novel transcript of SMYD3 was found. Comparing this transcript sequence with the published genomic DNA sequence, this new transcript contained a 111bp sequence located between 9803-9913bp of 7th intron of the SMYD3 which can be transcripted as an extron. This finding indicated that the alternative splicing patten of SMYD3 has no individual specificity and tissue specificity, as only one transcript mode was found among different oocytes and tissues including liver, lung, testis, kidney and heart.2. Effects of SMYD3-siRNA injection on bovine oocyte maturation, fertilization and embryo developmentThree siRNAs targeting three different regions (281-299, 567-585 and 1030-1048bp) of SMYD3 mRNA are designed and produced. Denuded oocytes (DOs) were generated by removing cumulus cells from Cumulus oocyte complexs (COCs) by mechan-pipetting at GV. The interference efficiency of injecting DOs with three SMYD3-siRNAs together is remarkable, as the relative expression quantity of SMYD3 mRNA decreased to 18% of that of no injection group. The results of injecting DOs with SMYD3-siRNAs showed that: there was no significant difference on maturation rate between SMYD3-siRNA injection and Nos-siRNA (nonsense siRNA)injection (67.14% vs 66.06%, P>0.05); and no significant differerce between the no injection groups of COCs and DOs(75.54% vs 76.19%, P>0.05); but the maturation rate of injection groups was remarkably lower compared with no injection groups(67.14%, 66.06% vs 75.54%, 76.19%, P<0.01). These results indicated that the mRNA level of SMYD3 had no effect on oocyte maturation in vitro, removing cumulus cells had no effect either, and the injury during injection may affect oocyte maturation.The oocytes of SMYD3-siRNA and Nos-siRNA injection groups and no injection groups of COCs and DOs were cultued in vitro for 22 hours and then fertilized. The results showed that there was no significant difference on cleavage rate among SMYD3-siRNA injection group, no injection group DOs and Nos-siRNA injection group(44.07% vs 42.8% vs 42.95%, P>0.05), but the rates of 8-cell and blastocyst were significantly lower in SMYD3-siRNA injection group than no injection group DOs and Nos-siRNA injection group(3.29% vs 17.51%, 12.82%; 0% vs 10.89%, 8.33%, P<0.01). This consequence showed that, compared with no injection group DOs, Nos-siRNA injection group, the SMYD3-siRNA injection group had no effects on fertilization and cleavage, but significantly decreased the rates of 8-cell and blastocyst. It is assumed that SMYD3 may activate some important genes which play key roles in embryo development from 2-cell stage to blastocyst. And there were no remarkable difference of the rates of 8-cell and blastocyst between no injection group DOs and Nos-siRNA injection group(17.51% vs 12.82%, 10.89% vs 8.33%, P>0.05). Consequently, the injury of microinjection had no influence on the results. Furthermore, the rates of cleavage, 8-cell and blastocyst of no injection COCs were significantly higher than other three groups(66.95% vs 42.8%, 42.95%, 44.07% ; 40.34% vs 17.51%, 12.82%, 29%; 30.47% vs 10.89%, 8.33%, 0%, P<0.01),which confirmed that cumulus cells are essential for fertilization.Fertilized oocytes injected SMYD3-siRNA and Nos-siRNA, and normal fertilized oocytes were cultured respectively. The results showed that there was no remarkable difference between the rates of cleavage, 8-cell and blastocyst among the SMYD3-siRNA, Nos-siRNA injection group and normal fertilized oocytes (55.88% vs 56.88% vs 58.43%; 32.35% vs 34.94% vs 31.90 %; 23.83% vs 29.0% vs24.01%, P>0.05). It suggested that influence of SMYD3 on affecting bovine embryo development from 2-cell stage to blastocyst is established during advanced stage of maturation rather than after fertilization.In short, knocking down SMYD3 mRNA of DOs had no effect on bovine oocyte maturation, fertilization and cleavage, however, decreased the development potential obviously. And there were no influence on cleavage and embryo development by knocking down the SMYD3 mRNA of zygotes 7 hours post fertilization. Therefore, it is hypothesized that the SMYD3 may activate some significant genes in the development from 2-cell stage to blastocyte, and this regulatory mechanism is established during advanced stage of maturation rather than after fertilization.This study can set a foundation for investigating the regulatory mechanism of SMYD3 during ooctye maturation, fertilization and embryo development.