Hamm Jennifer, Tessanne Kim, Murphy Clifton N, Prather Randall S
Division of Animal Sciences, University of Missouri, Columbia, Missouri.
Mol Reprod Dev. 2014 Jun;81(6):552-66. doi: 10.1002/mrd.22324. Epub 2014 Apr 16.
In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos.
体外胚胎生产对于动物繁殖、胚胎移植、转基因和克隆研究至关重要。然而,体外受精(IVF)胚胎通常发育延迟,且不如体内来源(IVV)的胚胎;这种差异可能是基因表达异常所致。通过定量PCR测定了在IVF、体细胞核移植(SCNT)、孤雌生殖和IVV猪卵母细胞及胚胎中,三个被认为在正常着床前胚胎发育中起重要作用的基因TRIM28、SETDB1和TP53的转录情况。体外成熟与体内成熟的卵母细胞之间,TRIM28或SETDB1的丰度没有差异(P>0.05),而体外成熟卵母细胞中的TP53水平更高。在IVF胚胎中,TRIM28从MII期卵母细胞增加到4细胞期和囊胚期,而IVV胚胎在整个发育过程中从成熟阶段开始TRIM28丰度降低。在所有处理组中,TP53的相对丰度在囊胚期增加,但IVF胚胎中的TP53相对丰度高于IVV和SCNT胚胎。相比之下,在所有处理中,SETDB1转录水平从2细胞期到囊胚期降低。对于每个分析的基因,难克隆和易克隆细胞系的SCNT胚胎与IVV胚胎的相似性高于IVF胚胎。敲低TRIM28对囊胚发育或SETDB1或TP53的表达也没有影响。因此,TRIM28、SETDB1和TP53在猪卵母细胞和胚胎中动态表达。此外,IVV和SCNT胚胎中TRIM28和TP53的丰度相似,但与IVF胚胎中的量不同。