Knockdown Of Dlg1 Has Effect On Porcine Oocytes Development In Vitro

The tumor suppressor ‘discs large homologue1 ’protein(Dlg1),is one of a family of protsins termed MAGUKs(membrane-associated guanylate kinase homologs).Dlg1 is involved in microtubules polarization、centrosome reorientation and cell migration through PDZ domain within Dlg1 interaction with other molecule.Mutation of Dlg1 results in a loss of cell polarity caused by abnormal assembly of microfilament and microtubule and some cell overproliferation.It has reported that mouse oocytes meiotic maturation induced the polarization of Dlgl and this polarized action is relevant to cytoskeleton.Porcine oocytes were used to determine the expression 、 location of Dlg1 during the process of oocytes maturation and early embryonic development.Methods of RNAi were used to further investigate the function in both cases.In this research,we examined the expression、localization and function of Dlg1 during porcine oocyte meiosis and early embryonic development.The quantitative RT-PCR(qRT-PCR)showed that Dlg1 is expressed in various tissues and cells.The results are shown below:1.Through the QRT-PCR detected the mRNA expression level of Dlg1 in a variety of porcine cells.In the result,it was highest level of Dlg1 mRNA in ovarian tissue and lung tissue,and lowest level in PEF cell.2、Immunofluorescence staining of Dlg1 in porcine oocytes and early embryos at different stages.Results showed that at the germinal vesicle(GV)stage,Dlg1 diffused to the cytoplasm,then clustered to the center,and finally,uniformly distributed in the cytoplasm in addition to the region of sub-cortex;at the early embryos satges,Dlg1 dispersed to the cytoplasm,but there was a continuous decline through development of embryo after parthenogenesis activation,the fluorescence level compared to the quantity of M Ⅱ oocytes declined by 46.9% 、 58.4% 、 29.66% in the 2-cells,4-cells and blastocyst embryos,respectively.3、Then we microinjected siRNAs into porcine GV oocytes respectively to screen a valid candidate sequence.Fluorescence microscopy observation and QRT-PCR demonstated thatsiRNA-2 had a higher efficiency,which recuded the Dlg1 expression 82.1% and 61.28% in m RNA and protein level respectively.4、Moreover,after injecting the Dlg1 siRNA-2 to the GV stage oocytes,the rates of PBE1(the first polar body extrusion)(25.2%±3.3%)were significantly reduced compared with the control(73.4% ± 4.7%).The assembly and formation of spindle as determined by immunofluorescent staining were abnormal,which were absent to control group.However,the percentage of cleavage and blastocyst were almost the same.The results in this study indicated that Dlg1 plays an essential role during porcine oocyte maturation but not early embryonic development.