Effects Of WT1Down-regulation On Oocytes And Preimplantation Embryo Development In Pigs

The Wilm’s tumour1gene is fundamental for the formation of the mouse andhuman urogenital systems. According to the studies of mRNA expression of WT1during development in the mouse and human suggest WT1is considered as atissue-specific gene with temporal and spatial variation in expression levels duringdevelopment, restricted to the urogenital system, mesenchymal structures and centralnervous system, thymus, heart, liver and spleen. Algaret and Moore et al. reported thatthe function of the WT1protein revealed that it is involved in celluar growth,proliferation, development, migration and survival. The WT1knockout mice sufferfrom abnormalities of the heart and mesothelial structures and failure of the gonadsand kidney. And the WT1knockout mice embryos show a higher degree ofprogrammed cell death, showing intrauterine death and a median survival time about14.5days in utero. Most likely, the failure of proper organogenesis depends, which onenhanced apoptosis.Recently, although most studies have focused on the potential role of WT1in theregulation of tumor in human and development of the gonads in mice, few researchershave investigated its expression characteristics and biological functions during oocyteand perimplantation embryonic development in pig. Pig is an attractive, large animalmodel for the study of certain human diseases. Herein, we examined the possibility ofan earlier role for WT1in controlling cell fate decisions using pLV3-WT1shRNA thatblock the WT1mRNA present during the perimplantation period. We evaluated WT1mRNA expression and WT1protein localization in oocyte and preimplantationembryos development. This study thus provides new insight on early embryogenesiswhich is relevant for future development biology studies. The main results wereshowed as follows:1. Comparison of the two methods: IVF and ICSIIn this study, we assessed three methods of optimization of porcine sperm(swimming-up method, single layer percoll method and double layer percoll method).The cleavage rats of sigle-and double-layer percoll method were significantly higher （60.8%and66.0%）than swimming-up method (48.8%)（P <0.05）. The blastocyst(32.5%) rates of the sperm dealing with double layer percoll method weresignificantly higher compare to that of swimming-up（15.3%）and single layer percollmethods（25.3%）during IVF （P <0.05）. In conclusion, optimization of porcinesperm with the double layer percoll method may be the most optimal method in IVF.We assessed IVF and ICSI methods, the results showed that in ICSI zygotes thepercentage of dual prokaryotic was higher than that of in IVF zygote (P <0.05),indicating that IVF might cause polyspermy. After fertilization, the cleavage rat andblastocyst rate were not big different between the two groups (P>0.05), but thenumber of cells in each blastocyst of ICSI was higher than IVF (P <0.05). Aftercomprehensive assessment of IVF and ICSI, we decided to choose ICSI embryos asour research objects.2. Expression of WT1in oocytes and preimplantation embryosCOCs with more than three layers of intact and compact cumulus cells wereselected, washed three times in manipulation fluid and cultured in in vitro maturation(IVM) medium. A group of about30COCs were cultured in a100ul of maturationmedium drop for42–44h at38.5℃in an atmosphere of5%CO2and saturatedhumidity. The mature oocytes were then divided into two groups: one group of matureoocytes to prepare for the post-detection; another group of mature oocytes to preparefor ICSI. Motile spermatozoa with normal morphology were selected from the doublelayer Percoll method. After ICSI, the zygotes were cultured in PZM-3medium at38.5℃in an atmosphere of5%CO2.RT-PCR and quantitative real-time PCR results showed that WT1mRNAexpressed in both pig oocytes and preimplantation embryos. WT1mRNA level peakedat the eight-cell and morula stages. WT1protein expression patterns were examined inoocytes and preimplantation embryos by immunofluorescence. And the expressionpattern in cytoplasm was stronger than nucleus.3. Construction a pLV3-WT1shRNA vector and the effects of WT1down-regulationon oocyte maturationWe have designed four shRNA sequences corresponding to WT1mRNA. TheWT1shRNA vectors were transfected into the porcine kidney fibroblasts (PKFs) toidentify which the vector was the most efficient shRNA in WT1down-regulation. According to the results of Real-time PCR and Western blot, shRNA-1100wasdetermined. The lentivairal vector with shRNA-1100(pLV3-WT1shRNA) wasconstructed and microinjected into the perivitelline space of the GV stage oocytes. Toevaluate the efficiency of the WT1knockdown, WT1mRNA expression wasexamined by quantitative real-time PCR. At MII stage, WT1expression wassignificant decreased compared with control oocytes (P <0.05). But the number ofvisible first polar body and glutathione (GSH) levels was not different between WT1down-regulation and control groups. From these results we could find that WT1down-regulation did not affect oocyte maturation.4. Effects of WT1down-regulation on embryo developmentTo further evaluate the function of WT1in porcine preimplantation embryonicdevelopment, pLV3-WT1shRNA was microinjected into the zygotes. The cleavagerate of in vitro cultured embryos was not different between the control and WT1down-regulation groups. On day5,59.2%and56.5%of the embryos from the controland Nc-shRNA groups had reached the blastocyst stage respectively. However, in theWT1down-regulation group, only9.2%of the embryos had reached the blastocyststage. Most of the WT1down-regulation embryos were arrested at the eight-cell (28%)and morula (32.2%) stage. Trypan blue uptake assay has been performed on day4, toexamine the lethality phenotype associated with WT1-shRNA microinjection.Approximately84%of the WT1down-regulation embryos contained some necrotic(blue-stained) cells.5. Effects of WT1down-regulation on apoptosis and growth-related genesThe number of apoptotic cells was estimated by a TUNEL assay performed onday5. We found that the number of apoptotic cells in WT1down-regulationblastocysts was higher than that of normal and Nc-shRNA embryos. Anti-apoptoticgene BCL2and apoptotic gene BAX mRNA level were examined using quantitativereal-time PCR at various developmental stages. The WT1down-regulation groupshowed a lower BCL2mRNA level at every developmental stage than controls(normal and Nc-shRNA), but BAX mRNA level was higher than control groups.