Gene Expression And Cloning Of Stem-loop Binding Protein During Pig Oocyte Maturation In Vitro

In oogenesis, translation adjustment plays a key role on gene expression translation. The mRNAs which are synthesised and stored in oocytes growth phase can be activated translation in late mature oocytes or early embryos. Although mRNAs encoding the histone proteins are among the most abundant in mammalian oocytes, the mechanism regulating their translation has not been fully understood. Replication-dependent histone mRNAs do not have a poly(A) tail but end instead in a conserved stem-loop structure and stem-loop binding protein is combinated on the stem-loop structure and plays an important role on stability and translation of the mRNAs. This accumulation of histone needs SLBP expressed during oocyte growth phase and stimulated translation at the high level during meiosis maturation. For this reason, taking the SLBP gene as candidate gene, expression and structure of the SLBP gene in porcine oocytes are studied in the current study.Oocyte maturation evaluationPorcine oocytes are stained by orcein to analysis nuclear phase. The results indicated that oocytes from3～6follicle was mainly in GV. Maturation24h in vitro, oocytes of91.5%had occurred GVBD. Maturation42h in vitro, oocytes of88.5%completed the first time meiotic, were holded in MII Maturation Oh,24h and42h in vitro, oocytes were in GV phase, GVBD phase and MII, respectively.Gene expression of SLBPThe results of RT-PCR demonstrated that SLBP mRNA was relatively expressed0.25,0.275,0.28,0.27and0.31in0h,12h,24h,36h and42h oocytes, respectively. From oocyte GV to MII periods, the level of mRNA expression SLBP was relative steady and the expression of each point had no significant differences.The results of Western Blotting demonstrated that the relative expression of SLBP protein were0.73,0.81and1.51in Oh,24h and42h, respectively and increased gradually. The highest expression of SLBP protein was in MII periods. Compared with GV and GVBD period, it had a significant difference(P<0.01). By comparing the protein maker, the apparent molecular weight of pig SLBP was41kDa. Maturation42h in vitro, the SLBP protein had an electrophoretic mobility shift and was presumed that SLBP protein was phosphorylated.SLBP cloning and bioinformatics analysisTaking SLBP cDNA conservative region of human and bovine as a template, five pairs of primers were designed and successfully amplified out porcine SLBP cDNA sequence for1053bp. The sequence covered the whole pig SLBP coding region was identified by NCBI-BLAST. The coding region was full-length828bp and started as ATG codon, terminated for TAA. The coding region encoded275-amino acid residuess. In these six kinds of mammals, SLBP coding sequence had the highest homology between pig and cow (90.7%), dog (82.73%), mice (81.64%), rat (81.16%), human (80.64%) and chimpanzee (80.31%). Through the sequence alignment, a sequence similar to CPE structure was identified in pig SLBP mRNA3’untranslated region.Research significanceThe finding of this study suggested that SLBP gene expression during oocyte maturation was stage-specific, so it indicated the SLBP had important relations with the stage of oocyte maturation. The transcription product in oocyte growth phase may regulated oocyte maturation and early embryo development. Maternal informations were responsible for completing meiosis and reconstruction during the whole process of mature oocytes to embryonic cells. These results verifed that the expression of SLBP protein in oocytes is different from the somatic cells. After pigs’SLBP gene coding region cloned and sequenced, new ideas for studying important molecular identification of the oocyte maturation quality, improving the oocytes quality and establishing efficient pig somatic cell nuclear transfer system were provided. From a macroscopic perspective, the improvement of oocyte maturation quality would make cloned pigs show greater advantages and application potential in the human animal disease models and xenotransplantation.