Studies Related To Factors Affecting Somatic Cell Nuclear Transfer In Pigs

To further enhance the efficiency of somatic cell nuclear transfer, and improve somatic cell nuclear transfer technical procedures of porcine, related factors include donor cell cycle regulation, in vitro maturation of recipient oocytes, in vitro culturation of reconstructed embryos, and somatic cells re-programming and other aspects s were systematically studied, and achieved the following results.1. Porcine fibroblast cell cycle regulation methods detected by flow cytometry is studied. In this study, we examined the effects of serum starvation, culture to confluence on cell cycle characteristics of porcine flbroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytomertry.24-72h serum starvation increased (P<0.05) the proportion of cells at the G0/G1phase (92.2-93.7%) as compared to the control group (77.8%). A similar increase in the percentage of Go/Gj were obtained in culturing to confluence group (94.4%and89.6%). The serum starvation cells were fully reversible with10%FBS after serum deprivation24h. While use these fibroblast synchronized by serum deprivation or culturing to confluence as donor to SCNT, there is no significant on reconstructed embryos on cleavage rate and blastocysts yield. In conclusion, porcine fibroblasts were effectively synchronized at G0/G1stages of the cell cycle by serum starvation or culturing to confluence, and there have no different on reconstructed embryos development while used as porcine SCNT donors.2. We investigated the effects of oocytes in vitro maturation duration treated with Activin A on nuclear maturation and cytoplasmic maturation. Oocytes were cultured for40h in TCM-199medium with or without Activin A treatment. The rates of first polar body emission were significantly increased compared with control group while oocytes matured in vitro treated with lOng/ml or100ng/ml Activin A (60.5%,66.1%vs.50.5%, P<0.05), but no significant different was found in the rates of bastocyst formation between treated or untreated with Activin A duration in vitro maturation while culturing parthenogenetic embryos. Mitochondrial distribution, a marker for cytoplasmic maturation, was revealed by using Mitotracker Red staining and confocal laser scanning microscopy. Oocytes exhibited changes in mitochondrial organization between treated and untreated with Activin A. Oocytes number were dramatical significantly increased with pattern C (diffused)(87.9%vs.66.4%, P<0.01) and dramatically decreased pattern A (peripheral)(1.8%vs.18.1%, P<0.01) and pattern B (semiperipheral)(8.4%vs.13.8%, P<0.05) distribution. This study demonstrated that duration in vitro maturation treated with Activin A can improve oocytes nuclear maturation and cytoplasmic maturation.3. Current NCSU-23was widely used as culture systems for porcine embryos and it is sub-optimal. The objectives of this study were to evaluate whether PZM-3or NCSU-23better support porcine embryo development. Briefly, porcine presumptive zygotes were produced after parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT) and cultured either in NCSU-23or PZM-3culture media. Transcript levels of genes involved in apoptosis (BCL-2), imprinted gene (Insulin-like growth factor2receptor,1GF2R), and metabolism (phosphoglycerate kinase1, PGK1) from SCNT blastocysts were detected by using Real-time PCR. Results showed that both cleavage and blastocyst rates have no signifcant different cultured in PZM-3or NCSU-23with PA and SCNT zygotes. However, we observed higher quality PA embryos in PZM-3compared with NCSU-23, increased the average blastocyst cell number in PA blastocyst (55.2±6.34vs.36.6±2.82, P<0.05) while it was no significant different in SCNT blastocyst (51.6±2.7vs.48.2±3.0,P>0.05). BCL-2and PGK1transcript levels were similar in blastocysts cultured in both PZM-3and NCSU-23media, but PZM-3media increased relative abundances of IGF2R mRNA in SCNT blastocysts compared to NCSU-23-derived blastocysts. Result showed that PZM-3medium better supported porcine early embryonic development than that of NCSU-23medium, and different culture media affected in vitro development and gene expression profile of embryos.4. Scriptaid, is a novel inhibitor of histone deacetylase, potentially enhancing cloning efficiency. In this study, we tested the effects of the HDAC inhibitor Scriptaid on preimplantation development and on histone acetylation and the gene expression of porcine SCNT embryos. The results showed that treated with500nM Scriptaid for12hours after reconstitution resulted in embryos (NT-S) that reached the blastocyst stage at a higher level (21.0%vs.9.7%, P<0.05) than that of the control (NT) embryos. In addition, unlike the NT embryos, the Scriptaid treated embryos displayed a global acetylated histone H3at lysine18profile similar to the in vitro-fertilized (IVF) embryos during the preimplantation development. We also determined several transcription factors of pluripotency gene OCT4and REXO1, the related histone acetylation gene HDAC2and HAT1, and the metabolism gene PGK1. The NT blastocysts showed similar levels of OCT4and PGK1as those of IVF blastocysts. However, the NT-S blastocysts showed similar levels of REXO1、HDAC2、HAT1and PGK1as those of IVF blastocysts, whereas the NT blastocysts showed significantly lower levels of REXO1and HAT1and higher level of HDAC2than those of IVF or NT-S blastocysts. Our data suggest that Scriptaid improves porcine SCNT preimplantation embryo development and affects the acetylated status of H3K18, rendering acetylation levels similar to those of the IVF counterparts.