Chemical activation of parthenogenetic and nuclear transfer porcine oocytes using ionomycin and strontium chloride

Che, Limei; Lalonde, A.; Bordignon, Vilceu

doi:10.1016/j.theriogenology.2007.02.006

Cited by 39 publications

(31 citation statements)

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“…Previous studies have shown that activation of oocytes with IONCSr 2C result in superior development of SCNT porcine embryos compared with activation with ION alone (Che et al 2007). One possibility is that Sr 2C treatment promotes a higher physiological response in the oocytes and consequently reduces the detrimental effects of cell cycle incompatibilities between the transferred nucleus and the host cytoplasm.…”

Section: H2ax139ph In Porcine Embryosmentioning

confidence: 99%

“…The reconstructed oocytes were activated using the following protocols (Che et al 2007): i) ION: the oocytes were exposed for 5 min to 15 mM ION in TCM-199 and then transferred to PZM-3 medium supplemented with CB (7.5 g/ml) and cycloheximide (CHX; 10 g/ml) for 4 h.…”

Section: Oocyte Activationmentioning

confidence: 99%

“…Strontium chloride (Sr 2C ) has been shown to induce calcium oscillations in oocytes, and it has, consequently, been used for oocyte activation in different species such as mouse, rat, bovine, and ovine species (Kline & Kline 1992, Kishikawa et al 1999, Méo et al 2004, Tomashov-Matar et al 2005, Zhang et al 2005, Choi et al 2013. We have previously shown that porcine oocyte activation using a combination of ionomycin (ION) and Sr 2C results in superior development of SCNT embryos compared with oocyte activation using ION alone (Che et al 2007).…”

Section: Introductionmentioning

confidence: 99%

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Phosphorylated histone H2A.x in porcine embryos produced by IVF and somatic cell nuclear transfer

et al. 2013

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Phosphorylated histone H2A.x (H2AX139ph) is a key factor for the repair of DNA double-strand breaks (DSBs) and the presence of H2AX139ph foci indicates the sites of DSBs. In this study, we characterized the presence of H2AX139ph during in vitro development of porcine embryos produced by IVF and somatic cell nuclear transfer (SCNT). Pronuclear stage embryos produced by IVF had, on average, 9.2 H2AX139ph foci per pronucleus. The number of H2AX139ph foci was higher in the 2-cell-stage embryos than in the 4-cell-stage embryos fixed at 48 h post-fertilization. The percentage of H2AX139ph-positive nuclei was higher in SCNT embryos that were activated with ionomycin (ION) alone than in those activated with ION and strontium chloride (IONCSr 2C ). A negative correlation was found between the percentage of H2AX139ph-positive cells and the total number of cells per embryo in day 7 blastocysts produced by IVF or SCNT. Based on the detection of H2AX139ph foci, the findings of this study indicate that DSBs occur in a high proportion of porcine embryos produced by either IVF or SCNT; fast-cleaving embryos have fewer DSBs than slow-cleaving embryos; the oocyte activation protocol can affect DNA integrity in SCNT embryos; and better-quality blastocysts have fewer DSBs. We propose that the presence of H2AX139ph foci can be a useful marker of embryo quality.

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Section: H2ax139ph In Porcine Embryosmentioning

confidence: 99%

Section: Oocyte Activationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Phosphorylated histone H2A.x in porcine embryos produced by IVF and somatic cell nuclear transfer

et al. 2013

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“…Under in vitro conditions, various artificial stimuli increase the intracellular calcium levels, for example, electric pulses (Sun et al, 1992;Koo et al, 2008), calcium ionophore Che et al, 2007) and chemicals that modulate the intracellular turnover of Ca 21 2002) and are used for the parthenogenetic activation of porcine oocytes.…”

Section: Introductionmentioning

confidence: 99%

“…The enzymes released from the CGs into the perivitelline space modify the zona pellucida to prevent additional sperm penetration (Bleil et al, 1981; reviewed by Williams, 2002). Calcium oscillations also induce cyclin B degradation and cytostatic factor inactivation, allowing the female chromatin entry into anaphase II (Lorca et al, 1993).Under in vitro conditions, various artificial stimuli increase the intracellular calcium levels, for example, electric pulses (Sun et al, 1992;Koo et al, 2008), calcium ionophore Che et al, 2007) and chemicals that modulate the intracellular turnover of Ca 21 2002) and are used for the parthenogenetic activation of porcine oocytes.Protein kinase C (PKC) is one of the transducers of the calcium signal system. Several studies have demonstrated the possible involvement of PKC in the oocyte activation process (Colonna et al, 1997;Green et al, 1999;Sedmíková et al, 2006) and the exocytosis of CGs (Bement and Capco, 1989;Fan et al, 2002).…”

mentioning

confidence: 99%

The effect of protein kinase C activator and nitric oxide donor on oocyte activation and cortical granule exocytosis in porcine eggs

Tůmová

Romar

Petr

et al. 2013

Animal

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Nitric oxide (NO) and protein kinase C (PKC) are involved in the activation of mammalian oocytes, although their role in the exit from the metaphase II stage and cortical granule (CG) exocytosis is still not fully understood. The aim of this study was to verify whether the NO-donor together with specific PKC-activators induce the complete activation of porcine oocytes assessed as meiosis resumption and a cortical reaction. Pig maturated oocytes were treated with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or PKC-activators such as phorbol-12-myristate-13-acetate (PMA, 100 nM), 1-oleoyl-2-acetyl-sn-glycerol (OAG, 400 mM) and L-a-phosphatidylinositol-3,4,5-trisphosphate dipalmitoyl heptaammonium salt (DPAM, 2 mM). To study the combined effect of NO-donor and PKC-activators, aliquots of oocytes were also incubated with SNAP (0.5 mM) together with PKC-activators at the same concentration as above (SNAP-DPAM, SNAP-OAG and SNAP-PMA groups). After in vitro maturation, an aliquot of oocytes was placed in a fresh medium without NO-donor or PKC-activators (Control group). Another aliquot of oocytes was activated by calcium ionophore A23187 (25 mM, 5 min). The results showed that 0% of the control oocytes reassumed meiosis. However, both the PKC-activators (DPAM 44.0 6 10.0%, OAG 63.3 6 1.0% and PMA 45.0 6 16.5%) as well as the NO-donor alone (48.7 6 21.0%) significantly induced exit from MII. Interestingly, the combination of PKC-activators and SNAP mainly restrained to the meiosis resumption (SNAP-OAG 0, SNAP-DPAM 17.4 6 2.5% and SNAP-PMA 38.4 6 8.5%). Control oocytes did not show a cortical reaction and the area occupied by CG reached 25.9 6 1.7%, whereas CGs were partially released after Ca 21 ionophore treatment (13.0 6 3.2%). Treatment with PKC-activators induced a cortical reaction compared with the control group (8.6 6 2.5, 6.7 6 1.9 and 0.7 6 0.4%, respectively, for DPAM, OAG and PMA groups). However, treatment with the NO-donor alone (SNAP group 17.2 6 2.2%) or combined with any PKC-activator prevented cortical reaction (SNAP-DPAM 20.7 6 2.6%, SNAP-OAG 16.7 6 2.9% or SNAP-PMA 20.0 6 2.4%). Besides, meiosis resumption was not always accompanied by a cortical reaction, indicating that these two activation events are independent. In conclusion, PKC-activators alone induce CG exocytosis to the same degree as calcium ionophore. However, an NO-donor alone or combined with PKC-activators is not able to induce a cortical reaction in pig oocytes.Keywords: protein kinase C, nitric oxide, pig oocyte, activation, cortical granules ImplicationsParthenogenetic activation of mammalian oocytes is commonly used in reproductive biotechnologies, for example, cloning using nuclear transfer, and its success depends on an adequate artificial stimulus. Nitric oxide (NO) has been described as an oocyte activator but it does not induce a cortical reaction. Protein kinases C (PKC) play an important role in the cortical reaction but the PKC cascade is not triggered after oocyte activation by an NO-donor. There is a possibilit...

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Presence of cleaved caspase 3 in swine embryos of different developmental capacities produced by parthenogenetic activation

Coutinho

Assumpção

Bordignon

2011

Molecular Reproduction Devel

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This study assessed the presence of cleaved caspase 3 (CC3) during the in vitro development of swine embryos produced by parthenogenetic activation (PA). Embryos with high and low capacity to develop into blastocysts and the exposure to a caspase inhibitor (z-DEVD-fmk) were used to investigate the effect of CC3 on embryo development. The blastocyst rate (64.3% vs. 16.4%) and the average number of nuclei per blastocyst (39.7 vs. 19.8) were significantly higher (P < 0.05) in early- (before 24 hr) compared to late- (between 24 and 48 hr) cleaving embryos after PA. CC3 was mainly detected in the cytoplasm of Day-2 and -4 embryos, but was primarily localized in the nucleus of Day-5 and -6 embryos. The fluorescence signal for CC3 relative to negative controls was significantly higher (P < 0.05) in early- (2.42-fold) compared to late-cleaving (1.39-fold) embryos at Day 2 of culture. Treatment with z-DEVD-fmk during the first 24 or 48 hr of the culture period resulted in more embryos developing into blastocysts compared to the control group (55.8% and 55.1% vs. 37%, respectively; P < 0.05). This study confirmed the presence of CC3 in PA embryos from the two-cell to the blastocyst stage, and revealed that CC3 cellular-localization changed during embryo development. CC3 was shown to be more abundant in early-cleaving and more developmentally competent embryos compared to late-cleaving and less developmentally competent embryos. The inhibition of caspase activity at the beginning, but not at the end, of the culture period affected development of PA embryos.

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Chemical activation of parthenogenetic and nuclear transfer porcine oocytes using ionomycin and strontium chloride

Cited by 39 publications

References 37 publications

Phosphorylated histone H2A.x in porcine embryos produced by IVF and somatic cell nuclear transfer

Phosphorylated histone H2A.x in porcine embryos produced by IVF and somatic cell nuclear transfer

The effect of protein kinase C activator and nitric oxide donor on oocyte activation and cortical granule exocytosis in porcine eggs

Presence of cleaved caspase 3 in swine embryos of different developmental capacities produced by parthenogenetic activation

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