Comparison of the RNA Polymerase I-, II- and III-Dependent Transcript Levels Between Nuclear Transfer and In Vitro Fertilized Embryos at the Blastocyst Stage

Abstract

Abstract. Cloned animals have been produced in several mammalian species so far, although success rates to term are very low. Aberrations in gene expression derived from abnormal epigenetic status have been thought to be a cause of developmental abnormalities in clones, and several abnormalities in gene expression have already been detected in cloned animals and embryos. In this study, we examined the hypothesis that the poor survival rates of nuclear transfer (NT) embryos are partly due to aberrations in the regulation of expression of genes transcribed by RNA polymerases I and III, in addition to polymerase II. We produced cloned and in vitro fertilized mouse embryos that developed to the blastocyst stage, and the amounts of several genes were analyzed using individual embryos. We found that the amounts of mature 18S ribosomal RNA (rRNA) transcribed by RNA polymerase I were lower in NT embryos than in IVF embryos, but that the amounts of 47S rRNA and intermediates of mature rRNAs were higher in NT embryos. In addition, the amount of 7SK RNA transcribed by RNA polymerase III was lower in NT embryos than in IVF embryos. The transcripts of all but one of the genes transcribed by RNA polymerase II were not noticeably different between NT and IVF embryos. These results suggest that some of the transcripts produced by RNA polymerases I, II and III are aberrantly regulated in NT embryos.

Sperm is epigenetically programmed to regulate gene transcription in embryos

“…Lastly, we have verified that the synthesis of rRNAs is not affected in spermatid-derived embryos. Indeed, in mouse, defect in rRNA synthesis has been proposed to explain the developmental defect of nuclear transfer embryos (Suzuki et al 2007;Zheng et al 2012) and could explain the difference in the bulk of RNA synthesis observed between sperm-and spermatid-derived embryos (Bui et al 2011). We observed that newly synthesized 18S and 28S rRNAs are equally well produced in sperm-and spermatid-derived embryos (Supplemental Fig.…”

“…Several hypotheses were proposed to explain the nature of this programming, including the idea that sperm is programmed for efficient replication after fertilization (Lemaitre et al 2005) or for supporting proper embryonic transcription (Suzuki et al 2007;Hammoud et al 2009;Zheng et al 2012). The latter hypothesis was proposed following the observation that promoters of developmentally important genes escape global replacement of histones by protamines in mature sperm.…”

“…The latter hypothesis was proposed following the observation that promoters of developmentally important genes escape global replacement of histones by protamines in mature sperm. In fact, these promoters retain post-translationally modified histones, suggesting that epigenetic marks on sperm chromatin may be transmitted to the embryo at fertilization and could subsequently pattern transcription of embryonic genes (Suzuki et al 2007;Hammoud et al 2009;Brykczynska et al 2010;Wu et al 2011;Paradowska et al 2012;Zheng et al 2012;Ihara et al 2014;Siklenka et al 2015). However, the validity of this hypothesis was recently questioned (Carone et al 2014;Samans et al 2014).…”

rRNA Genes Are Not Fully Activated in Mouse Somatic Cell Nuclear Transfer Embryos

“…This phenomenon has been reported previously between mouse in vitro fertilization and tail tip cell NT embryos. The author believed that, in NT blastocyst, the accumulation unprocessed 47 S rRNA and rRNA intermediates failed to be processed into mature rRNAs (31). Because no significant differences were found in FBL and B23 expressions between ICSI and NT groups in our study, the exact mechanism might be more complicated and need further investigations.…”

“…qPCR reactions were performed based on 1.5 l of cDNA sample, 10 l of TransStart TM Top Green qPCR SuperMix (TransGen, AQ131), and gene-specific primers (supplemental Table 1) in a 20-l reaction system on CFX96 Realtime System (Bio-Rad). H2afz, Hprt1, and Exno TM were used as candidate references for embryos (31,32). HP1␥, cyclophilin-A, and cyclophilin-B were used as candidate references for cells (13).…”