A Simplified Approach for Oocyte Enucleation in Mammalian Cloning

Iuso, Domenico; Czernik, Marta; Zacchini, Federica; Ptak, Grazyna; Loi, Pasqualino

doi:10.1089/cell.2013.0051

Cited by 25 publications

(21 citation statements)

References 23 publications

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“…Enucleate oocytes were injected with a nucleus, either from CTR or a Prm1-RFP fibroblasts suspended in PBS with 6% polyvinylpyrrolidone (Sigma-Aldrich). Activation and culture of reconstructed oocytes were processed as described ( Ptak et al., 2002 , Iuso et al., 2013a ). Qualities of obtained blastocysts were performed as described by Iuso et al.…”

Section: Methodsmentioning

confidence: 99%

Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures

et al. 2015

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SummaryProtamines confer a compact structure to the genome of male gametes. Here, we find that somatic cells can be remodeled by transient expression of protamine 1 (Prm1). Ectopically expressed Prm1 forms scattered foci in the nuclei of fibroblasts, which coalescence into spermatid-like structures, concomitant with a loss of histones and a reprogramming barrier, H3 lysine 9 methylation. Protaminized nuclei injected into enucleated oocytes efficiently underwent protamine to maternal histone TH2B exchange and developed into normal blastocyst stage embryos in vitro. Altogether, our findings present a model to study male-specific chromatin remodeling, which can be exploited for the improvement of somatic cell nuclear transfer.

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Section: Methodsmentioning

confidence: 99%

Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures

et al. 2015

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“…Somatic cell nuclear transfer (SCNT) is an effective method of reprogramming differentiated nuclei and has many potential applications (1). Owing to incomplete or faulty reprogramming, however, the cloning efficiency of SCNT embryos remains low (2,3).…”

Section: Introductionmentioning

confidence: 99%

IWP2 impairs the development of porcine somatic cell nuclear transfer embryos via Wnt signaling pathway inactivation

Huang

Yuan

et al. 2017

Biomedical Reports

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Abstract. Wnt signaling is critical in embryonic development and post-embryonic tissue homeostasis. The aim of the present study was to evaluate the expression levels of canonical Wnt signaling genes in porcine somatic cell nuclear transfer (SCNT) embryos. Quantitative polymerase chain reaction analysis was performed in porcine SCNT embryos, and the results indicated that the temporal expression patterns of canonical signaling genes were similar between in vivo and SCNT embryos from the 2-cell to the blastocyst stage. In addition, aberrant expression in a small number of Wnt signaling genes in SCNT embryos was identified. IWP2, an inhibitor of Wnt processing, was applied to the culture of SCNT embryos. The Wnt signaling pathway in the SCNT blastocysts may be inactivated via IWP2 treatment, reflecting the low expression levels of c-Myc and peroxisome proliferator-activated receptor δ. Furthermore, blastocyst hatching was damaged by IWP2 treatment. These findings indicate that the canonical Wnt signaling pathway is important for SCNT embryo development. IntroductionSomatic cell nuclear transfer (SCNT) is an effective method of reprogramming differentiated nuclei and has many potential applications (1). Owing to incomplete or faulty reprogramming, however, the cloning efficiency of SCNT embryos remains low (2,3). Aberrant reprogramming leads to changes in the expression levels of certain genes important for embryonic development. Studies on the expression of development-associated genes may be valuable for improving the developmental competence of SCNT embryos.Wnt signaling is a critical regulator of many cellular and physiological processes. Wnt signaling regulates cell proliferation and differentiation, controls migration and patterning during embryonic development, and maintains tissue homeostasis in adults (4,5). Depending on the involvement of β-catenin, these signaling pathways have been classified as canonical (β-catenin-dependent) or non-canonical (β-catenin-independent) (6). In the canonical Wnt signaling pathway, which has been better examined (7), β-catenin accumulates in the cytoplasm and eventually translocates to the nucleus where it acts as a transcriptional co-activator of transcription factors belonging to the T-cell factor/lymphoid enhancer factor-1(TCF/LEF) family. β-catenin that accumulates in the cytoplasm is degraded, however, by a protein complex that includes axin, glycogen synthase kinase-3 (GSK-3), protein phosphatase 2A (PP2A), casein kinase 1 (CK1) and adenomatous polyposis coli (APC) (6,8). This degradation complex becomes disrupted as soon as the Wnt protein binds to receptors of frizzled (Fz) and low-density lipoprotein receptor-related protein (LRP5/6), which activates multiple signaling cascades.Dysfunctions in Wnt signaling result in severe complications of embryonic development. For example, abnormalities were observed in the size and shape of mouse embryos lacking intact maternal/zygotic β-catenin during pre-and post-implantation development (5). By contrast, transient expression...

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“…SCNT was performed as previously described [24]. Briefly, mature sheep oocytes were incubated in Hepes-buffered TCM-199 medium containing 4 mg/ml BSA, 7.5 mg/ml Cytochalasin B and 5 mg/ml Hoechst 33342 in an incubator for 15 minutes.…”

Section: Methodsmentioning

confidence: 99%

Deregulated Expression of Mitochondrial Proteins Mfn2 and Bcnl3L in Placentae from Sheep Somatic Cell Nuclear Transfer (SCNT) Conceptuses

et al. 2017

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In various animal species, the main cause of pregnancy loss in conceptuses obtained by somatic cell nuclear transfer (SCNT) are placental abnormalities. Most abnormalities described in SCNT pregnancies (such as placentomegaly, reduced vascularisation, hypoplasia of trophoblastic epithelium) suggest that placental cell degeneration may be triggered by mitochondrial failure. We hypothesized that placental abnormalities of clones obtained by SCNT are related to mitochondrial dysfunction. To test this, early SCNT and control (CTR, from pregnancies obtained by in vitro fertilization) placentae were collected from pregnant ewes (at day 20 and 22 of gestation) and subjected to morphological, mRNA and protein analysis. Here, we demonstrated swollen and fragmented mitochondria and low expression of mitofusin 2 (Mfn2), the protein which plays a crucial role in mitochondrial functionality, in SCNT early placentae. Furthermore, reduced expression of the Bcnl3L/Nix protein, which plays a crucial role in selective elimination of damaged mitochondria, was observed and reflected by the accumulation of numerous damaged mitochondria in SCNT placental cells. Likely, this accumulation of damaged organelles led to uncontrolled apoptosis in SCNT placentae, as demonstrated by the high number of apoptotic bodies, fragmented cytoplasm, condensed chromatin, lack of integrity of the nuclear membrane and the perturbed mRNA expression of apoptotic genes (BCL2 and BAX). In conclusion, our data indicate that deregulated expression of Mfn2 and Bcnl3L is responsible for placental abnormalities in SCNT conceptuses. Our results suggest that some nuclear genes, that are involved in the regulation of mitochondrial function, do not work well and consequently this influence the function of mitochondria.

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A Simplified Approach for Oocyte Enucleation in Mammalian Cloning

Cited by 25 publications

References 23 publications

Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures

Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures

IWP2 impairs the development of porcine somatic cell nuclear transfer embryos via Wnt signaling pathway inactivation

Deregulated Expression of Mitochondrial Proteins Mfn2 and Bcnl3L in Placentae from Sheep Somatic Cell Nuclear Transfer (SCNT) Conceptuses

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