Dynamic Imaging of the Metaphase II Spindle and Maternal Chromosomesin Bovine Oocytes: Implications for Enucleation Efficiency Verification, Avoidanceof Parthenogenesis, and Successful Embryogenesis1

Dominko, Tanja; Chan, Anthony W.S.; Simerly, Calvin; Luetjens, C. Marc; Hewitson, Laura; Martinovich, Crista; Schatten, Gerald

doi:10.1095/biolreprod62.1.150

Cited by 77 publications

(56 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Efficient enucleation is a crucial step to avoid such problems as aneuploidy abnormalities and possibly parthenogenetic activation [33]. However, using a blind enucleation method could affect enucleation rate, which is influenced by a number of factors.…”

Section: Discussionmentioning

confidence: 99%

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

Ren

Lü

et al. 2010

J Assist Reprod Genet

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Ren

Lü

et al. 2010

J Assist Reprod Genet

View full text Add to dashboard Cite

show abstract

“…A problem associated with the procedure is that incomplete enucleation results in aneuploidy. This then leads to genetic interference within the recipient cytoplasm and abnormal development [61]. The removal of genetic content from the recipient cyotoplast also tends to delete factors that are important for nuclear reprogramming.…”

Section: Second Metaphase Stage Cytoplasts As Recipientsmentioning

confidence: 99%

Oocyte-associated transcription factors in reprogramming after somatic cell nuclear transfer: a review

Yin¹,

Liu²,

Bou³

et al. 2014

Front. Agr. Sci. Eng.

View full text Add to dashboard Cite

Oocytes are unique cells with the inherent capability to reprogram nuclei. The reprogramming of the somatic nucleus from its original cellular state to a totipotent state is essential for term development after somatic cell nuclear transfer. The nuclear-associated factors contained within oocytes are critical for normal fertilization by sperm or for somatic cell nuclear reprogramming. The chromatin of somatic nuclei can be reprogrammed by factors in the egg cytoplasm whose natural function is to reprogram sperm chromatin. The oocyte first obtains its reprogramming capability in the early fetal follicle, and then its capacity is enriched in the late growth phase and reaches its highest capability for reprogramming as fully-grown germinal vesicle oocytes. The cytoplasmic milieu most likely contains all of the specific transcription and/or reprogramming factors necessary for cellular reprogramming. Certain transcription factors in the cytoplast may be critical as has been demonstrated for induced pluripotent stem cells. The maternal pronucleus exerts a predominant, transcriptiondependent effect on embryo cytofragmentation, with a lesser effect imposed by the ooplasm and the paternal pronucleus. With deep analysis of transcriptomics in oocytes and early developmental stage embryos more maternal transcription factors inducing cellular reprogramming will be identified.

show abstract

“…However, this approach adversely affects viability of cells and future embryos (7). The alternative solution is blind enucleation when metaphase chromosomes are recognized by location of the first polar body that serves as a reference point.…”

mentioning

confidence: 99%

Optimization of Parameters of the Oocyte Enucleation and Fusion With the Somatic Cell During Production of Mammalian Cloned Embryos

Сингина¹,

Лопухов²,

Zinovieva³

et al. 2013

S-h. biol.

View full text Add to dashboard Cite

S u m m a r yParameters of the oocyte enucleation and fusion with somatic cell for producing of cattle cloned cetohybrids were studied. It was established that the electrofusion efficiency depends on strength, duration and set number of electrical pulse. The optimal parameters for Multiporator (Eppendorf, Germany) were determined: two consistent pulses DC electrical field each of 30 V field strength and 15 msec duration followed by the pulse of AC electrical field of 5 V and 10 sec duration. It was found that the first polar body serving as a marker for the chromosome position at «blind» enucleation must not deviate from the chromosome location by more than 10 degrees.Keywords: in vitro culture, oocyte, cattle nuclear transfer, electrofusion, enucleation.Somatic cloning is widely used in biomedical research and in production of transgenic animals (1). Despite a certain success of using this technique in recent years, it is yet inefficient in cattle and other domestic animals owing to high incidence of embryonic abnormalities and low viability of the born cloned offspring (2-4).To create a clone, genetic material of the host's egg cell is replaced by genetic material of a somatic cell (reprogramming). It is often associated with complete removal of the oocyte's chromosomes at metaphase II, after which the somatic donor cell is introduced in the perivitelline space of the (5).The procedure is visualized by staining of the chromosomes with fluorescent vital dye Hoechst 33342 allowing enucleation of the host oocyte under ultraviolet light (6). However, this approach adversely affects viability of cells and future embryos (7). The alternative solution is blind enucleation when metaphase chromosomes are recognized by location of the first polar body that serves as a reference point. This method is considered as more safe for viability of the egg cells and their capacity to future embryonic development (8), though some problems are not excluded too. After isolation of the first spindle pole body, metaphase plate of chromosomes stays for a while in a close proximity to it. Then, with aging of the egg cell, chromosomes disconnect from the spindle pole body that migrates to the perivitelline space. This circumstance complicates blind enucleation of oocytes or interdicts it (9), which necessitates identifying the factors important for efficiency of the method.After enucleation, the fusion of cytoplast and karyoplast is performed to form a new reconstructed egg cell. Following development of cloned embryos directly depends on successful association of the host and donor cells, and viability of the resulting hybrid (10-14).Electrofusion is commonly used technique for karyoplast-cytoplast fusion. A pulsed electric field applied to the cell membrane causes in it a reversible loss of integrity -transmembrane pores, after which the membrane becomes compressed and discharged. This makes possible the fusion of two contacting membranes, whose contact points open cytoplasmic channels for exchange of cytoplasmic colloid. As a result,...

show abstract

Dynamic Imaging of the Metaphase II Spindle and Maternal Chromosomesin Bovine Oocytes: Implications for Enucleation Efficiency Verification, Avoidanceof Parthenogenesis, and Successful Embryogenesis1

Cited by 77 publications

References 21 publications

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

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

Oocyte-associated transcription factors in reprogramming after somatic cell nuclear transfer: a review

Optimization of Parameters of the Oocyte Enucleation and Fusion With the Somatic Cell During Production of Mammalian Cloned Embryos

Contact Info

Product

Resources

About