Identification and Characterization of an Oocyte Factor Required for Porcine Nuclear Reprogramming

Kong, Qingran; Xie, Bingteng; Li, Jingyu; Ye, Huan; Huang, Tianqing; Wei, Renyue; Liu, Zhong Hua

doi:10.1074/jbc.m113.543793

Cited by 20 publications

(19 citation statements)

References 61 publications

(58 reference statements)

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“…After injection, oocytes were kept for at least 2 h before manipulations, which allows the antibody to bind endogenous REST. Moreover, 10 picoliters of 10 M REST-LNA (Locked Nucleic Acid, Exiqon) was injected into porcine oocytes with the first polar body collected at 33 h of IVM (35), and the oocytes matured at 42 h were used for NT, ICSI, and IVF. The procedure for porcine NT, PA, and IVF has been described previously (35).…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, 10 picoliters of 10 M REST-LNA (Locked Nucleic Acid, Exiqon) was injected into porcine oocytes with the first polar body collected at 33 h of IVM (35), and the oocytes matured at 42 h were used for NT, ICSI, and IVF. The procedure for porcine NT, PA, and IVF has been described previously (35). After fusion, 0.1 M SB431542 was used to treat NT embryos for 12 h. Cumulus cell-free oocytes were directly activated by the same parameters as for the somatic cell nuclear transfer procedure to produce PA embryos.…”

Section: Methodsmentioning

confidence: 99%

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RE1-silencing Transcription Factor (REST) Is Required for Nuclear Reprogramming by Inhibiting Transforming Growth Factor β Signaling Pathway

Kong

Xie

Zhang

et al. 2016

Journal of Biological Chemistry

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Edited by Eric R. FearonDifferentiated cells can be reprogrammed by transcription factors, and these factors that are responsible for successful reprogramming need to be further identified. Here, we show that the neuronal repressor RE1-silencing transcription factor (REST) is rich in porcine oocytes and requires for nuclear transfer (NT)-mediated reprogramming through inhibiting TGF␤ signaling pathway. REST was dramatically degraded after oocyte activation, but the residual REST was incorporated into the transferred donor nuclei during reprogramming in NT embryos. Inhibition of REST function in oocytes compromised the development of NT embryos but not that of IVF and PA embryos. Bioinformation analysis of putative targets of REST indicated that REST might function on reprogramming in NT embryos by inhibiting TGF␤ pathway. Further results showed that the developmental failure of REST-inhibited NT embryos could be rescued by treatment of SB431542, an inhibitor of TGF␤ pathway. Thus, REST is a newly discovered transcription factor that is required for NT-mediated nuclear reprogramming.Embryonic cells differentiate into all three germ layers of the body as development progresses. Once differentiated, the reversion of the differentiated state to pluripotency is strictly limited in normal development. However, experimentally the differentiated state can be returned to the pluripotent state by transcription factors (1, 2). Despite numerous attempts, the factors responsible for successful nuclear reprogramming still need to elucidate. Transcription factors maintaining the pluripotency of embryonic stem cells (ESCs) 3 are called pluripotent factors, and they have an important role in nuclear reprogramming, such as Oct4, Sox2, and Nanog (3, 4). Thus, we can identify and characterize reprogramming factors by screening the pluripotent factors. The repressor element 1 (RE1)-silencing transcription factor (REST), as a zinc finger protein, binds 21-bp RE1 sites and functions as a key negative regulator of neurogenesis, so it is also called neuron-restrictive silencer element (5). Recently, REST has been reported to induce gene expression by recruiting TET3 to the DNA for directed 5hmC generation and Nuclear SET domain-containing protein 3-mediated H3K36 trimethylation in neurons (6). Furthermore, REST has different roles in different cellular contexts, such as oncogenic and tumor-supressor functions and hematopoietic and cardiac differentiation (7,8). In 2008, REST was proved to maintain self-renewal and pluripotency of mouse ESCs through suppression of microRNAs and believed to be a major pluripotent factor (9, 10). However, it has not been elaborated in nuclear reprogramming. Here, we provide evidence that REST plays a unique role in NT-mediated reprogramming as a supressor of the TGF␤ signaling pathway in pig. ResultsExpression Pattern of REST-We first investigated the expression of REST in porcine oocytes, nuclear transfer (NT), and parthenogenetic activation (PA) embryos by real-time PCR and Western blotting analysis. P...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

RE1-silencing Transcription Factor (REST) Is Required for Nuclear Reprogramming by Inhibiting Transforming Growth Factor β Signaling Pathway

Kong

Xie

Zhang

et al. 2016

Journal of Biological Chemistry

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“…Aspirated oocytes with an evenly granulated cytoplasm and at least three uniform layers of compact cumulus cells were selected and washed three times with maturation medium (TCM199 (Invitrogen) plus 0.05 mg mL À1 epidermal growth factor, 0.5 mg mL À1 LH and 0.5 mg mL À1 FSH (all Sigma-Aldrich)). The oocytes with or without microinjection were cultured in 24-well plates (Corning) containing 500 mL of maturation medium at 398Cin 5% CO 2 in air and saturated humidity (Kong et al 2014).…”

Section: Methodsmentioning

confidence: 99%

“…Each experiment was repeated three times and at least 30 oocytes were examined each time. In addition, the same instrument settings were used for each replicate (Xu et al 2009;Kong et al 2014).…”

Section: Western Blotmentioning

confidence: 99%

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Endo-siRNA deficiency results in oocyte maturation failure and apoptosis in porcine oocytes

Liu¹,

Zhao²,

Piao³

et al. 2017

Reprod. Fertil. Dev.

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Abstract. Both microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs) play key regulatory roles in gene expression. Some studies have demonstrated that the function of miRNA is suppressed in mouse oocytes, suggesting that endo-siRNA, not miRNA, is essential for female meiosis. This finding has yet to be confirmed in other species. In this study, by knockdown of DICER1, DROSHA and its cofactor DiGeorge syndrome critical region 8 (DGCR8) in porcine oocytes, we found that the proportion of oocytes with DICER1 deficiency that developed to meiosis II (MII) stage was significantly lower than oocytes with DROSHA and DGCR8 deficiency (39.23 versus 68.71 and 71.25% respectively; P , 0.05). Oocytes lacking DROSHA and DGCR8 formed a barrel-shaped metaphase I spindle, with chromosomes tightly aligned at the metaphase plate whereas most oocytes (87%) lacking DICER1 showed spindle abnormalities during oocyte in vitro maturation. Furthermore, DICER1 deficiency also resulted in oocyte apoptosis. These results indicate that endo-siRNAs are essential for oocyte maturation in pigs.

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The Oocyte Determinants of Early Reprogramming

Schwarzer

Boiani

2014

Epigenetic Mechanisms in Cellular Reprogramming

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The oocyte is the female germ cell specialized for processing the sperm genome as well as the only cell in the adult body that can convert, i.e., reprogram, the genome of a donor somatic cell from a differentiated to a totipotent state. One of the big open questions in this field pertains to the identity of the natural components of the oocyte that can achieve nuclear reprogramming. We would like to call them determinants of reprogramming. In our view the experimental pursuit of these determinants must be preceded by a review of the oocyte's properties. These can be ascribed to qualitative and quantitative traits such as size, nuclear architecture, cytoplasm-to-nucleus ratio, and molecular makeup of the oocyte. In addition, the oocyte's ability to achieve fast and full reprogramming suggests that the nuclear and/or cytoplasmic molecules in charge of the reprogramming process are abundant. We hypothesize that the reason for such abundance may be simple: these molecules are normally used to process the sperm genome upon fertilization and are repurposed for reprogramming. Among these molecules, maternal-effect factors including transcription factors and chromatin remodeling factors may prime the reprogramming process and determine its initial speed. Here, we discuss known and putative factors involved in reprogramming, such as DJ-1, Brg1, Oct4, Glis1, and Tctp1, that were identified by candidate gene, transcriptomic, and proteomic approaches. Shedding light on the natural network of reprogramming factors found in the oocyte will help reveal general principles of cell rejuvenation for the benefit of aging studies and regenerative medicine.

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Identification and Characterization of an Oocyte Factor Required for Porcine Nuclear Reprogramming

Cited by 20 publications

References 61 publications

RE1-silencing Transcription Factor (REST) Is Required for Nuclear Reprogramming by Inhibiting Transforming Growth Factor β Signaling Pathway

RE1-silencing Transcription Factor (REST) Is Required for Nuclear Reprogramming by Inhibiting Transforming Growth Factor β Signaling Pathway

Endo-siRNA deficiency results in oocyte maturation failure and apoptosis in porcine oocytes

The Oocyte Determinants of Early Reprogramming

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