Parthenogenetic Activation of Porcine Oocytes by Electric Pulse and/or Butyrolactone I Treatment

Dinnyés, András; Hirao, Yuji; Nagai, Takashi

doi:10.1089/15204559950019843

Cited by 18 publications

(21 citation statements)

References 43 publications

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“…One group received 2 direct current pulses of 100 V/mm for 20 sec in 0.28 mol/L mannitol supplemented with 0.1 mmol/L MgSO 4 , 0.05 mmol/L CaCl 2 , and 0.01% PVA, and then cultured in 5 g/ml of CB-supplemented medium for 4 h. The eggs in the other 3 groups were subjected to 2 direct current pulses and then cultured in media supplemented with either 50 M butyrolactone [15] (Funakoshi, Tokyo, Japan), 2 mmol/L 6-dimethylaminopurine (6-DMAP, Sigma) [16], or 10 g/ml cyclohexamide (Sigma) for 4 h [17]. Eggs in the fifth group were treated with 15 mol/L calcium ionomycin (Sigma) for 20 min followed by 2 mmol/L 6-DMAP for 4 h [11].…”

Section: Activation Of Nuclear-transferred Eggsmentioning

confidence: 99%

Production of Cloned Pigs from Adult Somatic Cells by Chemically Assisted Removal of Maternal Chromosomes1

Yin

Tani

Yonemura³

et al. 2002

Biology of Reproduction

203

175

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The present study demonstrated that brief treatment of in vitro-matured porcine oocytes with demecolcine results in a membrane protrusion that contains a condensed chromosome mass, which can be easily removed by aspiration. This simple, chemically assisted method for removing maternal chromosomes enabled the production of a large number of nuclear-transferred porcine eggs. The development of eggs whose chromosomes were removed by this procedure following transfer of somatic cell nuclei to the blastocyst stage was not significantly different among groups activated using different procedures (6% to 11%) and was also not different among donor cells of different origins (3% to 9%), except for cumulus cells (0.4%). After transfer of 180 to 341 nuclear-transferred eggs that received somatic cells to 6 recipients, 2 of the recipients produced 8 healthy cloned piglets from the heart cells of a female pig. The chemically assisted method for removing maternal chromosomes was also effective for bovine and rabbit eggs.

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Section: Activation Of Nuclear-transferred Eggsmentioning

confidence: 99%

Production of Cloned Pigs from Adult Somatic Cells by Chemically Assisted Removal of Maternal Chromosomes1

Yin

Tani

Yonemura³

et al. 2002

Biology of Reproduction

203

175

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“…Studies on uniparental embryos, such as gynogenotes, androgenotes, and parthenotes, are essential to our understanding of genome imprinting. Porcine parthenotes can be obtained by electrical stimulation of metaphase-II (M-II) oocytes (Hagen et al 1991, Prather et al 1991, Lee et al 2004, by the treatment with chemicals, such as ionophores (Hagen et al 1991, Wang et al 1998, or by a combination of electrical activation and treatment with chemicals, such as protein synthesis inhibitors (Nussbaum & Prather 1995), or protein kinase inhibitors (Dinnyes et al 1999). The most effective and widely used method of activating oocytes is electrical stimulation.…”

Section: Introductionmentioning

confidence: 99%

Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes

Somfai

Ozawa²,

Noguchi³

et al. 2006

Reproduction

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We investigated nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to cytochalasin B (CB) during in vitro maturation (IVM). Nuclear progression was similar in control oocytes and oocytes matured in the presence of 1 mg/ml CB (IVM-CB group) by 37 h IVM; at this time the proportion of oocytes that had reached or passed through the anaphase-I stage did not differ significantly between the IVM-CB and the control groups (61.3 and 69.9% respectively; P!0.05). After IVM for 37 h, no polar body extrusion was observed in the IVM-CB group. In these oocytes, the two lumps of homologous chromosomes remained in the ooplasm after their segregation and turned into two irregular sets of condensed chromosomes. By 41 h IVM, the double sets of chromosomes had reunited in 89.5% IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached the metaphase-II stage by this time. When IVM-CB oocytes cultured for 46 h were stimulated with an electrical pulse and subsequently cultured for 8 h without CB, 39.0% of them extruded a polar body and 82.9% of them had a female pronucleus. Chromosome analysis revealed that the majority of oocytes that extruded a polar body were diploid in both the control and the IVM-CB groups. However, the incidence of polyploidy in the IVM-CB group was higher than that in the control group (P!0.05). In vitro development of diploid parthenotes in the control and the IVM-CB groups was similar in terms of blastocyst formation rates (45.8 and 42.8% respectively), number of blastomeres (39.9 and 44.4 respectively), the percentage of dead cells (4.3 and 2.9% respectively), and the frequency of apoptotic cells (7.3 and 6.3% respectively). Tetraploid embryos had a lower blastocyst formation rate (25.5%) and number of cells (26.2); however, the proportion of apoptotic nuclei (7.0%) was similar to that in diploid parthenotes. These results suggest that the proportion of homozygous and heterozygous genes does not affect in vitro embryo development to the blastocyst stage.

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“…(A) Cattle oocytes were cultured in control medium for 0-44 h or in control medium for 24 h and then in BL I for 3-20 h, or 24 h in control medium and 5 h in BL I followed by transfer back to control medium for 3-15 h, and at the indicated time intervals the samples were collected for histone H1 and MBP double kinase assay by adding both substrates in a kinase cocktail. While bohemine treatment alone had only small potential for activating bovine 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 54 M. Kubelka et al MII oocytes (Alberio et al, 2000), BL I at a concentration of 150-200 µM was shown to be able to activate pig oocytes at quite high rates (Dinnyes et al, 2000). Ten oocytes were used for each sample in both assays.…”

Section: Discussionmentioning

confidence: 99%

“…As mentioned earlier, several papers have reported activation of unaged pig or cattle oocytes by a combination of different stimuli, such as ionomycin/ionophore, ethanol or electric pulse combined with 6-DMAP or cycloheximide (Presicce & Yang, 1994;Tanaka & Kana-Figure 3 Histone H1 kinase and MAP kinase activities in control and BL I-treated oocytes. For that reason, more recently, specific inhibitors of cdk kinases (bohemine and BL I) have been used in combination with ionomycin (Alberio et al, 2000) or with electric pulse (Dinnyes et al, 2000) for activation of cattle and pig oocytes, respectively. (B) Changes in phosphorylation status of two MAPKs (ERK1 and ERK2) as detected by mobility shifts in samples from control and BL I-treated oocytes shown by western blotting.…”

Section: Discussionmentioning

confidence: 99%

“…(B) Changes in phosphorylation status of two MAPKs (ERK1 and ERK2) as detected by mobility shifts in samples from control and BL I-treated oocytes shown by western blotting. Dinnyes et al (2000) also showed that the combination of BL I with an electric pulse did not significantly increase the rates of activation; however, the nuclear configuration was significantly changed: while activation by BL I alone resulted in 13-20% of parthenotes forming a haploid pronucleus and two polar bodies and 47-53% of parthenotes forming two pronuclei without extrusion of the second polar body, the combination of BL I with an electric pulse induced the formation of two pronuclei and one polar body in the great majority of the parthenotes obtained. The experiment was conducted three times and a representative example is shown.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Activation of pig and cattle oocytes by butyrolactone I: morphological and biochemical study

Kubelka

Anger

Pavlok

et al. 2002

Zygote

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In this study a specific inhibitor of cyclin-dependent kinases (cdks), butyrolactone I (BL I), was used for activation of pig and cattle metaphase II (MII) oocytes. BL I at a concentration of 100 μM was able to induce activation of both pig and cattle MII oocytes in a manner dependent on exposure time; however, precise timing of BL I exposure was required for the best activation results. The optimum activation rates were obtained when cattle MII oocytes were treated for 5 h with BL I and subsequently for 3-11 h in control medium, and pig MII oocytes for 8 h in BL I and then for 8-16 h in control medium; the percentage of activated oocytes after such treatment varied between 55% and 74% and between 53% and 81% for cattle and pig oocytes, respectively. Shorter exposures to BL I led to re-entry of the oocytes to the metaphase state in 35-50% of oocytes, the remaining oocytes forming a pronuclear stage; longer exposure to BL I led to increased numbers of oocytes being abnormal or degenerated. The behaviour of histone H1 kinase and mitogen activated protein (MAP) kinase, also measured during the experiment, reflected the morphological changes in the oocytes: both were inactivated after BL I treatment, though the inactivation of histone H1 kinase occurred 2 h ahead of that of MAP kinase. However, in the oocytes treated for a shorter time with BL I, with the reoccurrence of condensed chromatin in proportion of the oocytes cultured in control medium after BL I treatment, both kinases became reactivated. Taken together, these results suggest the possibility of using BL I for activation and cloning experiments in both species.

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Parthenogenetic Activation of Porcine Oocytes by Electric Pulse and/or Butyrolactone I Treatment

Cited by 18 publications

References 43 publications

Production of Cloned Pigs from Adult Somatic Cells by Chemically Assisted Removal of Maternal Chromosomes1

Production of Cloned Pigs from Adult Somatic Cells by Chemically Assisted Removal of Maternal Chromosomes1

Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes

Activation of pig and cattle oocytes by butyrolactone I: morphological and biochemical study

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