Earticle

초록

영어

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nuclei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2’deoxycytidine (5-aza-dC), DNA methylation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molecular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptotic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-related genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

목차

ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
  In Vitro Porcine Oocyte Maturation and Parthenogenic Activation
  Donor Ear Fibroblast Culture and Preparation
  Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Assay
  Nuclear Transfer
  Terminal Deoxynucleotidyl Transferasemediated dUTP Nick-End Labeling (TUNEL) Assay
  Immunofluorescence Staining
  Immunostaining and Quantification of Fluorescence Intensity
  Real Time Reverse Transcription Polymerase Chain Reaction(Real Time RT-PCR)
  Trichostatin A (TSA) Treatment
  5-aza-2’-deoxycytidine (5-aza-dC) Treatment
  Statistical Analysis
 RESULTS
  Effect of TSA and 5-aza-dC Treatment on Porcine SCNT Embryos Viability
  Effect of TSA and 5-aza-dC on Blastocyst Cell Number and Programmed Cell Deth of Porcine Nucleus Transfer Embryos
  Quantification Via Real-Time RT-PCR of the mRNA ExpressionProgrammed Cell Deth (PCD) in Porcine Blastocysts
  Effect of TSA and 5-aza-dC Treatment on the Acetlationand Methylation Status of Histon H3K9 at the Blastocysts
  Quantification Via Real-Time RT-PCR of the mRNA Expression DNA Methyltransferase (DNMT) in Porcine Blastocysts
 DISCUSSION
 REFERENCES

키워드

저자

• Sung Hyun Lee [ Department of Animal Sciences, Chungbuk National University, Cheongju 361-763, South Korea ]
• Yong-Nan Xu [ Department of Animal Sciences, Chungbuk National University, Cheongju 361-763, South Korea ]
• Young-Tae Heo [ Department of Animal Sciences, Chungbuk National University, Cheongju 361-763, South Korea ]
• Xiang-Shun Cui [ Department of Animal Sciences, Chungbuk National University, Cheongju 361-763, South Korea ]
• Nam-Hyung Kim [ Department of Animal Sciences, Chungbuk National University, Cheongju 361-763, South Korea ] Corresponding author

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  Reproductive & developmental biology

• 간기

  계간

• pISSN

  1738-2432

• eISSN

  2288-0151

• 수록기간

  1977~2018

• 십진분류

  KDC 527 DDC 636