Developmental Competence of HMC Derived Bovine Cloned Embryos Obtained from Somatic Cell Nuclear Transfer of Adult Fibroblasts and Granulosa Cells

Bhojwani, Sanjay; Vajta, Gábor; Callesen, Henrik; Roschlau, K.; Kuwer, Andreas; Becker, Frank; Alm, H.; Torner, H.; Kanitz, W.; Poehland, Ralf

doi:10.1262/jrd.17025

Cited by 18 publications

(12 citation statements)

References 43 publications

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“…In our previous studies [16,18], we demonstrated successful application of the HMC TM method in our laboratory. In spite of our continued success in carrying out NT experiments, we frequently encounter huge variations in blastocyst rates.…”

Section: Discussionmentioning

confidence: 88%

“…Both the classic procedure [2] and the still relatively new method of handmade cloning (HMC TM ) [23,16] have led to live offspring. However, both techniques are problematic because they require resetting of the gene expression program of somatic cells to that consistent with early embryonic development.…”

Section: Discussionmentioning

confidence: 99%

“…Nuclear transfer experiments were conducted using the HMC TM method established by Vajta et al [17] and described in detail by Bhojwani et al [16,18]. Briefly, oocytes were collected using the ovarian slicing method [21].…”

Section: Nuclear Transfer and Embryo Culturementioning

confidence: 99%

“…In our previous study [16], we found that the m e t h o d o f " h a n d m a d e c l o n i n g " ( H M C T M ) e s t a b l i s h e d b y V a j t a [ 1 7 ] i s s u i t a b l e f o r reconstructing a large number of embryos by NT. The fact that the obtained blastocyst rates of up to approximately 45% in relation to the number of reconstructed embryos varied greatly between different experiments led us to question whether the origin and culture conditions of the defined male and female fibroblast lines could be responsible for the differences in developmental potency.…”

mentioning

confidence: 99%

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Donor Cell Lines Considerably Affect the Outcome of Somatic Nuclear Transfer in the Case of Bovines

Poehland¹,

Al-Rostum²,

Becker³

et al. 2007

Journal of Reproduction and Development

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Abstract. Since the first successful nuclear transfer (NT) experiments were carried out, various somatic cell types have been used as donor cells for production of cloned animals. In most experiments, fibroblasts are used since they only need to be isolated and cultivated. Recently, some researchers have shown that different cell cultures from different sources possess different capacities to support preimplantation development of NT embryos. The blastocyst rates obtained in our previous studies varied and were as high as 45% in relation to the number of reconstructed embryos. This led us to question whether the origin and culture conditions of the defined male and female fibroblast lines could be responsible for the differences in developmental potency. Taking all our results into consideration, we conclude that different fibroblast lines recovered from the same tissue and cultivated under equal culture conditions could produce dramatically different blastocyst rates. The influence of cell line itself is higher than the influence of passage number. The observed effects of cell cycle stage, chromosomal aberrations, and diminished vitality are important but not sufficient to discriminate well-qualified nuclear donor cells. We speculate that some epigenetically regulated deviations in the gene expression program are responsible for these phenomena. Explanation of the underlying mechanisms should contribute to better understanding of epigenetic reprogramming and may ultimately assist reprogramming in the laboratory.

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Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Nuclear Transfer and Embryo Culturementioning

confidence: 99%

mentioning

confidence: 99%

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Donor Cell Lines Considerably Affect the Outcome of Somatic Nuclear Transfer in the Case of Bovines

Poehland¹,

Al-Rostum²,

Becker³

et al. 2007

Journal of Reproduction and Development

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“…The rate of development to blastocysts as well as the efficiency of birth of healthy animals is not different when embryos were produced by HMC compared with the conventional procedure (Bhojwani et al 2005, Lagutina et al 2007. Currently, several groups are using HMC as the first alternative to generate cloned animals from different species.…”

Section: Introductionmentioning

confidence: 99%

Effect of zona pellucida removal on early development of in vitro produced bovine embryos

et al. 2013

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SUMMARYDuring early embryo development the zona pellucida acts as a barrier against polyspermia and guarantees communication between blastomeres before and during compaction. However, the development of new technologies of embryo production such as "Handmade Cloning" demands removal of this membrane. The aim of this study was to determine the effect of zona pellucida removal on in vitro bovine embryo development. First, the consequences of zona pellucida removal was assessed by comparing the percentage of first cleavage, percentage of blastocysts and cell number among zona-included and zona-free embryos; either through the removal of the zona pellucida immediately after IVF or parthenogenesis. Embryo development was also evaluated when zona pellucida was removed before parthenogenesis. In a second set of experiments, the gene expression levels of BAX, BCL2, CASP3, CDH1, OCT4 and SOX2 were evaluated in zona-free and zona-included IVF-derived embryos. No significant differences were found in the percentage of first cleavage, percentage of blastocyst and cell number on IVF-embryos cultured with or without zona. Parthenogenetic embryos followed the same general pattern even when the zona pellucida was eliminated before activation; however there was a significant increase in the rate of first cleavage when the zona pellucida was removed after activation but this did not impact upon further development. Furthermore, no significant differences in gene expression level were found between zona-free and zona-included IVF-embryos for the studied genes. We concluded that the lack of zona pellucida did not affect the early development when an appropriate system is used for embryo culture to ensure blastomeric contact and normal compaction.

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Ultrastructural analysis reveals striking differences of intercellular contact lengths in bovine embryos produced in vivo, in vitro and by somatic cell nuclear transfer

Bhojwani

Tomek

Jonas

et al. 2006

Molecular Reproduction Devel

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Cellular coherence and communication, thus cell-to-cell contact is an indispensable premise to sustain the formation of complex, multi-cellular organisms. We have analyzed intercellular contact lengths in NT-cloned bovine embryos compared to the in vivo or in vitro produced counterparts. Therefore, ultrastructural analysis was carried out by transmission electron microscopy (TEM) at the 8-cell and blastocyst stage of development. To obtain embryos generated in vivo, oviducts of superovulated cows were flushed 3 days after insemination, subsequent to slaughter. Standard in vitro maturation (IVM) and -fertilization (IVF) were utilized to obtain in vitro embryos. Cloned embryos by somatic nuclear transfer were produced by the handmade cloning (HMC) procedure. The points of apposition/focal contact points (CPs) between the blastomeres were of the shortest order in cloned embryos (236 +/- 135 nm) and of highest order in the in vivo produced embryos (2,085 +/- 1,540 nm), although no significant differences regarding the blastomere sizes in the various groups of 8-cell embryos could be established. In summary, the CP lengths in case of in vitro and in vivo 8-cell embryos were, on an average, five or nine times longer, respectively, than in the case of the cloned embryos. These differences of CP lengths vanished in embryos reaching the blastocyst stage of embryonic development in all the three groups of embryos. The observed differences of intercellular contact length at distinct stages of embryonic development could be responsible for differences in intercellular communication between the blastomeres at the beginning of cellular differentiation. These may be one reason for the lower developmental competence of cloned (NT) embryos.

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Developmental Competence of HMC Derived Bovine Cloned Embryos Obtained from Somatic Cell Nuclear Transfer of Adult Fibroblasts and Granulosa Cells

Cited by 18 publications

References 43 publications

Donor Cell Lines Considerably Affect the Outcome of Somatic Nuclear Transfer in the Case of Bovines

Donor Cell Lines Considerably Affect the Outcome of Somatic Nuclear Transfer in the Case of Bovines

Effect of zona pellucida removal on early development of in vitro produced bovine embryos

Ultrastructural analysis reveals striking differences of intercellular contact lengths in bovine embryos produced in vivo, in vitro and by somatic cell nuclear transfer

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