Apoptosis and In Vitro Development of Preimplantation Porcine Embryos Derived In Vitro or by Nuclear Transfer1

Hao, Yanhong; Lai, Liangxue; Mao, Jiude; Im, Gi‐Sun; Bonk, Aaron J.; Prather, Randall S.

doi:10.1095/biolreprod.103.016170

Cited by 103 publications

(94 citation statements)

References 30 publications

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“…Considering that NT embryos are experimentally produced whereas fertilized embryos are made by the natural process, it is reasonable to think that transferred somatic nuclei undergo higher cellular stress than sperms. This notion is supported by the fact that NT embryos exhibit higher rates of apoptosis and weaker stress-coping functions than fertilized embryos (60)(61)(62). Nevertheless, the proportion of NT embryos that develop to the blastocyst stage is similar to that of in vitro fertilized embryos in many reports.…”

Section: Discussionmentioning

confidence: 84%

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Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

Miyamoto

Nagai²,

Kitamura³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the fourcell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.oocyte extract and proteomics | reprogramming in mammalian oocytes E mbryonic cells differentiate into specific types of cells as development progresses. Once differentiated, the reversion of a differentiated cell state to an original undifferentiated state is strictly inhibited in normal development. However, it has been experimentally shown that differentiated nuclei can be returned to an undifferentiated embryonic state after nuclear transfer (NT) to enucleated eggs or oocytes (1, 2). Such experiments provide an opportunity to reprogram somatic cells as a means to prepare undifferentiated cells, which may be differentiated into any kinds of cells for cell-replacement therapy. Recently, nuclear reprogramming technology has been expanded by the production of induced pluripotent stem (iPS) cells (3). iPS cells can be obtained by overexpressing specific sets of transcription factors such as Oct4, Sox2, Klf4, and c-myc in cultured cells. The processes leading to establishment of iPS cell lines are being carefully examined and we are begining to understand how somatic cells acquire pluripotency by this method (4-6). The mechanisms leading to pluripotency may be different between iPS cells and NT embryos because somatic nuclei transferred into unfertilized metaphase II (MII) oocytes must undergo early embryonic development before the inner cell mass (ICM) can give rise to pluripotent embryonic stem (ES) cells. In addition, the molecules and mechanisms that induce somatic cell reprogramming are expected to be different between iPS cells and NT embryos (7,8). A recent study has shown that nuclear t...

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Section: Discussionmentioning

confidence: 84%

“…NT embryos undergo higher rates of apoptosis (60) and are less tolerant to the in vitro culture environment than fertilized embryos (61). Lower levels of gene expression related to cell protection from stress and apoptosis have been reported in NT embryos, compared with fertilized embryos (62).…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

Miyamoto

Nagai²,

Kitamura³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage in individual eukaryotic as well as in preimplantation embryos, and can be used for the detailed analysis of DNA breaks during apoptosis [16]. Hao et al [15] used TUNEL assay on porcine SCNT embryos and found that the earliest positive TUNEL signals happened in blastocysts. In this study, comet assay was used to analyze apoptosis in porcine cloned embryos, showing that the damaged embryos containing apoptotic cells could be detected in all developmental stages before blastocyst.…”

Section: Discussionmentioning

confidence: 99%

“…The COC with uniform cytoplasm and at least three layers of compact cumulus cells were chosen and rinsed three times in Tyrode lactate-HEPES (TL-HEPES) containing 0.l% polyvinyl alcohol (PVA), then in vitro matured (IVM) as previously described by Wu et al [26]. Briefly, approximately [15][16][17][18][19][20] germinal vesicle (GV) stage oocytes were placed into a 100-μl droplet of pre-equilibrated culture medium (TCM199; GIBCO BRL, Gaithersburg, MD) supplemented with 10 IU/ ml PMSG (Tianjin Huafu High & New Biotech Co., Tianjin, China), 10 IU/ml hCG (Ningbo Hormonal Reagents Co., Ltd, Zhejiang, China), 69 μg/ml L-cysteine, 1 μg/ml 17β-estradiol, 10%(v/v) porcine follicular fluid (pFF, self-made), 36 mg/l sodium pyruvate, 1 ml/l sodium lactate, 10% newborn calf serum (NCS, GIBCO BRL), 70 mg/l penicillin G and 50 mg/l streptomycin, covered with mineral oil. According to the experimental design, the oocytes were cultured for various periods at 38.5°C in humidified atmosphere of 5% CO 2 in air.…”

Section: Methodsmentioning

confidence: 99%

“…Apoptosis was first observed in bovine SCNT embryos at the 4-cell stage, but for in vitro fertilization (IVF) embryos at 6-to 8-cell stages using TUNEL assay [14]. In pigs, the earliest positive TUNEL labeling signals were detected in SCNT-derived blastocysts, and the percentage of cells undergoing apoptosis in the SCNT embryos was apparently higher than that of IVF-derived blastocysts [15]. DNA damage and fragmentation are commonly major characteristics of apoptosis, and the comet assay (single cell gel-electrophoresis, SCGE) appears to be a more sensitive method for assessing DNA damage to evaluate the apoptosis individual eukaryotic cell as well as in mammalian preimplantation embryos, because this method is able to distinguish DNA fragmentation present in all apoptotic stages, including early ones [16,17].…”

Section: Introductionmentioning

confidence: 99%

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Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro

Ren

Lü

et al. 2010

J Assist Reprod Genet

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Purpose The purpose of this study was to investigate the relationship of porcine somatic cell nuclear transfer (SCNT) embryo developmental competence with embryonic cell apoptosis and DNA methylation. Methods The apoptotic incidence was examined via comet assay, and the mRNA expression of genes implicated in apoptosis (Bcl-2) and DNA methylation (Dnmt1, Dnmt3a) was determined using real-time RT-PCR. Results Comet assay showed that the SCNT embryos exhibited significantly higher apoptotic rate at 2-cell stage (8.3% versus 2.1%, P<0.05), 16-cell stage (27.3% versus 19.2%, P<0.05) and morula (37.5% versus 26.9, P<0.05) compared with IVF embryos. Compared with IVF embryos, a higher Bcl-2 mRNA expression pattern was observed in SCNT embryos before the 8-cell stage and differed significantly at 2-and 4-cell stages (P<0.05). After the 16-stage, Bcl-2 mRNA expression pattern became significantly lower in SCNT group (P <0.05). The relative expression level of Dnmt1 mRNA showed a higher expression level in oocytes, then sharply decreased and started to increase slightly after the 8-cell (IVF embryos) or 16-cell stage (SCNT embryos). Dnmt1 mRNA expression in IVF embryos appeared to have been lower than that of SCNT group before 16-cell stage embryos, especially at 4-and 8-cell stages (P<0.05). Although a trend for a similar increase of Dnmt3a expression was observed in IVF and SCNT embryos after 8-cell embryos, SCNT group resulted in much higher Dnmt3a mRNA abundance compared with the IVF group, particularly after 16-cell embryos (P<0.05).Conclusions The results showed that low efficiency of porcine SCNT technology may be associated with either embryonic apoptosis or incomplete reprogramming of donor nuclear caused by abnormal Dnmts mRNA expression.

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Differential gene expression patterns in porcine nuclear transfer embryos reconstructed with fetal fibroblasts and mesenchymal stem cells

Kumar

Jin

Kim

et al. 2006

Developmental Dynamics

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The present study compared the developmental ability and gene expression pattern at 4-cell, 8-cell, morula, and blastocyst stages of porcine nuclear transfer (NT) embryos from fetal fibroblasts (FFs) and mesenchymal stem cells (MSCs), in vitro fertilized (IVF), and in vivo derived embryos. MSC-NT embryos showed enhanced blastocyst formation, higher total cell number, and a low incidence of apoptosis compared to FF-NT embryos. Alterations in the expression pattern of genes implicated in transcription and pluripotency (Oct4, Stat3, Nanog), DNA methylation (Dnmt1, Dnmt3a), histone deacetylation (Hdac2), growth factor signaling, and imprinting (Igf2, Igf2r) and apoptosis (Bax, Bcl2) regulation were observed in NT embryos. The expression of transcripts in MSC-NT embryos more closely followed that of the in vivo derived embryos compared with FF-NT embryos. In conclusion, MSCs with a relatively undifferentiated genome might serve as suitable donors that could be more efficiently reprogrammed to re-activate expression of early embryonic genes in porcine NT.

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Apoptosis and In Vitro Development of Preimplantation Porcine Embryos Derived In Vitro or by Nuclear Transfer1

Cited by 103 publications

References 30 publications

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos

Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro

Differential gene expression patterns in porcine nuclear transfer embryos reconstructed with fetal fibroblasts and mesenchymal stem cells

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