Oxamflatin Treatment Enhances Cloned Porcine Embryo Development and Nuclear Reprogramming

Abstract

Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro-fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve cloning efficiency.

Expression of pluripotency‐related genes is highly dependent on trichostatin A‐assisted epigenomic modulation of porcine mesenchymal stem cells analysed for apoptosis and subsequently used for generating cloned embryos

“…On the one hand, those epigenetic modifiers represent not only the subclass of highly specific extrinsic HMT inhibitors (HMTi) such as G9A (H3K9) HMTi, whose pivotal member is diazepin‐quinazolin‐amine derivative termed BIX‐01294 (Cao et al, ; Huang et al, ), but also the subclass of ectopic non‐specific DNMT inhibitors, whose most important members are: (a) 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) (Huan, Wang, et al, ; Huan, Wu, et al, ; Huan et al, , ; Ning et al, ); and (b) zebularine (a nucleoside analog of cytidine; Diao et al, ). On the other hand, they represent the subclass of ectopic non‐selective HDAC inhibitors (HDACi), whose main members are (a) TSA (Huan, Wang, et al, ; Huan, Wu, et al, ; Huan et al, ; Opiela et al, ; Samiec et al, ); (b) scriptaid (SCPT) (Liang, Zhao, Choi, Kim, & Cui, ; Xu et al, ; Zhang et al, ); (c) oxamflatin (Hou et al, ; Mao et al, ); (d) sodium butyrate (NaBu) (Kumar et al, ; Liu et al, ); (e) m ‐carboxycinnamic acid bis hydroxamide (CBHA) (Song et al, ); (f) panobinostat, also known as LBH589 (Jin et al, ); (g) abexinostat, also termed PCI‐24781 (Jin et al, ); (h) quisinostat, also called JNJ‐26481585 (Jin, Guo, et al, ); and (i) dacinostat, also named as LAQ824 (Jin, Lee, Taweechaipaisankul, Kim, & Lee, ). The initiation of chromatin decondensation and gene transcriptional activity is triggered both via highly specific and transient inactivation of G9A (H3K9) HMTs by BIX‐01294 (Cao et al, ; Huang et al, ) and via non‐specifically blocking the biocatalytic activity of either DNMTs by 5‐aza‐dC and zebularine (Diao et al, ; Saini et al, ) or HDACs by TSA, SCPT, oxamflatin, NaBu, CBHA, panobinostat, abexinostat, quisinostat and dacinostat (Jin, Lee, et al, ; Jin et al, , ; Jin, Guo, et al, ; Kumar et al, ; Song et al, ; Zhang et al, ).…”

Generation of transgenic‐cloned Huanjiang Xiang pigs systemically expressing enhanced green fluorescent protein

“…For example, in our laboratory, the blastocyst rate of porcine SCNT embryos is often <20%, while the data from IVF embryos are usually as higher as 50% (Misumi et al, ). Large evidences indicate incompatible epigenetic modifications, including DNA methylation and histone acetylation, occurred during nuclear reprogramming in porcine SCNT embryos are closely associated with the low overall efficiency of pig cloning (Mao et al, ; Samiec, Opiela, Lipiński, & Romanek, ; Zhao, Whyte, & Prather, ). Several epigenetic‐regulating drugs, such as 5‐aza‐2′‐deoxycytidine, trichomycin A (TSA), scriptaid and oxamflatin, have been developed and applied to pig cloning, resulting in significant enhancement of the in vitro and in vivo developmental capacity of cloned porcine embryos (Mao et al, ; Samiec et al, ; Zhao et al, ).…”

Applications of omics and nanotechnology to improve pig embryo production in vitro

“…Also in the pig, treatment with Oxamflatin for 16 hr postactivation enhanced blastocyst formation of SCNT embryos and produced more live piglets than the Scriptaid group which was treated under the same conditions. Also, methylation profiles of POU5F1 regulatory elements and centromeric repeat elements were reduced in the Oxamflatin‐treated group when compared to the untreated control group (Mao et al, ). Oxamflatin treatment for 6 hr postfusion and activation increased blastocyst rates and total cell number in the blastocysts when compared to the untreated group.…”

“…Since establishment of embryonic stem cell (ESC) lines remains to be a challenge in pigs, SCNT is a commonly used method to produce cloned genetically modified pigs (Hou et al, ). However, due to inadequate nuclear reprogramming of donor cells, the cloning efficiency is still low, approximately 1–5% (Mao et al, ). Defects in DNA methylation and histone modifications have often been associated with reduced developmental potential in cloned embryos (Luo, Ju, Muneri, & Rui, ).…”

“…Searching for expression patterns more closely related to a normal in vivo‐derived embryo and the identification of aberrantly reprogrammed transcripts could allow for targeted correction of gene expression during the cloning process (Whitworth et al, ). Several studies evaluated the potential of histone deacetylase inhibitors (HDACi), such as 6‐(1,3‐dioxo‐1H, 3H‐benzo[de]isoquinolin‐2‐yl)‐hexanoic acid hydroxyamide (Scriptaid) (Whitworth et al, ; Zhang et al, ; Zhao et al, ; Zhao et al, ), suberoylanilide hydroxamic acid (SAHA), 4‐iodo‐SAHA (ISAHA) (Whitworth et al, ), (2E)‐5‐[3‐[(phenylsufonyl) aminol phenyl]‐pent‐2‐en‐4‐ynohydroxamic acid (Oxamflatin) (Mao et al, ; Park et al, ) and trichostatin A (TSA) (Huan et al, ; Zhao et al, ) to improve pig cloning efficiency. It is thought that hyperacetylation will enhance transcription and reprogramming by opening the chromatin structure and allowing RNA polymerase II to access transcription start sites more easily (Van Thuan et al, ).…”