Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide

Presicce, Giorgio Antonio; Yang, Xiangzhong

doi:10.1002/mrd.1080380405

Cited by 92 publications

(57 citation statements)

References 39 publications

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“…CCB and CCD have been widely used to prevent extrusion of pseudo PBs of G0/G1 somatic donors during the SCNT procedure [3]. With a similar purpose, exposure to these chemicals at different times, such as 1, 5 or 6 hr, have also been attempted in bovine [1,11,20,24,38]. In the present study, groups 1-4 showed extrusion rates ranging from 53-67%, whereas group 5 exhibited a significantly low rate of 22%.…”

Section: Discussionmentioning

confidence: 70%

“…Liu et al [19] reported 27% blastocyst rates using CHX combined with 2.5 µg/ml CCD for 6 hr after Ca 2+ ionophore treatment, but they could not obtain development to the hatched blastocyst stage. Presicce and Yang [24] also reported that the combination of CHX with CCB after ethanol treatment could improve the development of bovine parthenotes. Improved development after treatment with CCB or CCD may be due to their ability to induce diploid parthenotes by preventing cytokinesis, and formation of diploid embryos is similar to that of the normal fertilization process, thus, resulting in better developmental rates [19,24].…”

Section: Discussionmentioning

confidence: 99%

“…Although artificial stimuli, such as Ca 2+ ionophore [19], ionomycin [23], ethanol [31], electric pulse [27] and strontium (SrCl 2 ) [21] can activate bovine oocytes by increasing intracellular Ca 2+ , the efficiency of artificial stimuli is limited [25,34]. Therefore, additional chemical agents, such as cycloheximide (CHX, protein phosphorylation inhibitor) [24] and 6-dimethylaminopurine (DMAP, protein synthesis inhibitor) [29,30], that prevent MPF re-accumulation by reducing cdc2 kinase activity [2] have also been applied. DMAP treatment of metaphase II oocytes induces diploid activation by preventing chromosomal separation and extrusion of the second polar body (PB) [19].…”

mentioning

confidence: 99%

“…This implies the need to analyze the specific effect of chemical activation agents on the ploidy of embryos both at early and later stages instead of examining only at the blastocyst stage. Based on the above facts, the present study was designed to evaluate the effects of CCB with 5 hr CHX treatment after ionomycine treatment, based on commonly used methods [12,17,24], on the parthenogenetic development and ploidy of early and later stages of bovine embryos.…”

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confidence: 99%

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Parthenogenetic Development and Ploidy following Various Chemical Activation Regiments of Bovine Oocytes

Ock

Rho

2008

The Journal of Veterinary Medical Science

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ABSTRACT. Bovine oocytes treated using various combinations of 5 µM ionomycin (Ion), 10 µg/ml cycloheximide (CHX) and 5 µg/ml cytochalsin B (CCB) were evaluated to determine developmental rates and ploidy status. The groups were comprised of metaphase II oocytes exposed to Ion for 5 min (Group 1); Ion treatment followed by CHX for 5 hr (Group 2); Ion treatment followed by CHX/CCB for 1 hr and CHX for 4 hr (Group 3); Ion treatment followed by CHX/CCB for 3 hr and CHX for 2 hr (Group 4); and Ion treatment followed by CHX/CCB for 5 hr (Group 5). Group 5 exhibited significantly (P<0.05) lower rates of second polar body (PB) extrusion and higher rates of cleavage and blastocyst development. At 8 hr after Ion treatment, the eggs in groups 2, 3 and 5 were divided into 2 subgroups based on the presence or absence of the second PB and were assessed for cleavage rate and ploidy at the two-cell stage. The cleavage rates did not differ among the activation treatments or between the presence and absence of the second PB in all groups. The diploid rate was significantly (P<0.05) higher in group 5 than in groups 2 and 3. However, the diploid rate in blastocyst-stage parthenotes did not differ among groups 2, 3 and 5. Consequently, oocyte activation by CHX/CCB for 5 hr after Ion treatment could enhance diploid parthenogenetic development in bovine. KEY WORDS: bovine parthenote, chemical activation, ploidy, polar body.J. Vet. Med. Sci. 70(11): 1165-1172, 2008 In bovine, oocyte activation is a major factor for successful production of reconstructed embryos by somatic cell nuclear transfer (SCNT) [34], parthenogenesis and intracytoplasmic sperm injection [23]. During normal fertilization in mammalian species, sperm penetration into the oocyte triggers intracellular Ca 2+ oscillation followed by inactivation of the maturation promoting factor (MPF) composed of the catalytic subunit of cdc2p34 protein kinase and the regulatory subunit cyclin B1/B2 for about 2 hr [37] [29,30], that prevent MPF re-accumulation by reducing cdc2 kinase activity [2] have also been applied. DMAP treatment of metaphase II oocytes induces diploid activation by preventing chromosomal separation and extrusion of the second polar body (PB) [19]. However, CHX does not prevent chromosomal segregation and second PB extrusion, resulting in haploid development. When cytochalasin D (CCD) was added in the medium, chromosomal segregation manifested, but the cytokinesis that induces second PB extrusion was prohibited in CHX-treated oocytes, leading to diploid development with two pronuclei in activated oocytes [4,19]. The combined treatment of ionomycin with DMAP for oocyte activation could induce an abnormal pattern of karyokinesis during the first cell cycle [8], and thus, CHX has widely been used as an alternative approach [19]. Moreover, in cloning, oocyte activation using CHX produces healthy live offspring in cattle [12,17], but the live offspring rate is still low [5].Campbell et al. [3] proposed the hypothesis that when donor cells at G1 stage are reconstructed...

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Section: Discussionmentioning

confidence: 70%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Parthenogenetic Development and Ploidy following Various Chemical Activation Regiments of Bovine Oocytes

Ock

Rho

2008

The Journal of Veterinary Medical Science

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“…Activation is an important step in the NT procedure and ethanol and ionomycin have been widely used as activating agents in several species, such as cattle [15,20,21], mice [8], sheep [16], and goats [1,4,6,11,32]. There are a few reports on the development of goat oocytes after parthenogenetic activation by treatment with ionomycin and ethanol, both followed by exposure to 6-diethylaminopurine (6-DMAP) [17,18].…”

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confidence: 99%

The Effect of Activation Protocols on the Development of Cloned Goat Embryos

Apimeteetumrong

Thuangsanthia²,

Leingcharoen³

et al. 2004

The Journal of Veterinary Medical Science

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ABSTRACT. This study was conducted to compare the developmental competence of somatic nuclear transfer (NT) embryos, after either ionomycin or ethanol activation, in locally bred goats. Donor cells were prepared from the ear skin fibroblasts of a female goat. Cells, at passage 3-8, starved by culturing in 0.5% FCS for 4-8 d, were used for NT. Immature oocytes were obtained from FSH-stimulated goats and matured for 22 hr before enucleation and NT. After fusion, the reconstructed embryos were activated with either ionomycin or ethanol followed by culturing in 6-dimethylaminopurine (6-DMAP) and cytochalasin B (CB), for 3 hr. In experiment I, the fuse d NT embryos (n=63, ionomycin and n=68, ethanol treatments, respectively) were cultured in B 2 with a Vero co-culture system and their developmental competence was evaluated through to Day 9. In experiment II, the NT embryos at the 2-4 cell stage on Day 2 derived from each treatment (ionomycin n=46, and ethanol n=37), were transferred into 10 synchronous recipients. There were no significant differences between the NT embryos derived from the ionomycin and ethanol groups, in fusion (86.3% versus 82.9%), cleavage (90.5% versus 82.4%) and for morula/blastocyst development rates (9.5% versus 5.9%). Sixty percent (3/5) of the recipients from ionomycin became pregnant by midterm (2.5 mts) while only 20% (1/5) from ethanol treatment was pregnant by Day 45. The results demonstrate that activation with either ionomycin or ethanol in combination with 6-DMAP-CB treatment does not affect the development of cloned goat embryos. KEY WORDS: activation, developmental competence, goat, nuclear transfer.J. Vet. Med. Sci. 66(12): 1529-1534, 2004 Since the cloned sheep named "Dolly", was produced by transferring nuclei from adult cells into enucleated oocytes [27], there have been many studies carried out to produce other cloned mammals such as mice, cattle, goats, pigs and rabbits [1,5,19,25,26]. The nuclear transfer (NT) technique has been efficiently used for producing transgenic, cloned farm animal offspring. These animals are capable of producing valuable proteins, which could have a marked impact on the pharmaceutical industry [22] [1,4,6,11,32]. There are a few reports on the development of goat oocytes after parthenogenetic activation by treatment with ionomycin and ethanol, both followed by exposure to 6-diethylaminopurine (6-DMAP) [17,18]. Nevertheless, comparison of the development of NT goat embryos with either ionomycin or ethanol, as an activating agent, has not been reported. The objective of this study was to compare the developmental competence in vitro and in vivo of NT goat embryos, after activation, by using either ionomycin or ethanol, both followed by incubation in 6-DMAP-cytochalasin B (CB). MATERIALS AND METHODSUnless otherwise indicated, all chemicals used in this study were obtained from Sigma-Aldrich Company (St. Louis, MO) and the media from Gibco Invitrogen Corporation (Grand Island, NY).Preparation of donor cells: Donor cells were prepared from an ear skin f...

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Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation

Tateno

Kamiguchi

1997

Mol. Reprod. Dev.

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This study was undertaken parthenogenetically to activate Chinese hamster oocytes in vitro by chemical stimuli. Oocytes were exposed to five different chemical agents, ethanol (EtOH), strontium chloride (SrCl2), cycloheximide (CHX), phorbol ester (PMA), and ionophore A23187 (IA23). No parthenogenetic activation was observed in the oocytes treated with 8% EtOH for 8–11 min, 1.7 mM and 5.0 mM SrCl2 for 1 hr, 100 μM and 400 μM CHX for 2 hr, and 81 nM and 162 nM PMA for 5 min. In contrast, 89.7% of oocytes parthenogenetically extruded the second polar body in treatment with 3 μM IA23 for 5 min, but only 22.6% of them formed a pronucleus and developed to 2‐cell embryos. The remaining ova stopped their cell cycle immediately after completion of the second meiotic division. They had unichromatid chromosomes (monads), which are called MIII chromosomes. Treatment with 5 μM IA23 for 5 min was so deleterious that >90% of oocytes were degenerated. However, oocyte activation was significantly improved when the treatment with 3 μM IA23 for 5 min was followed by treatment with 8% EtOH for 10 min, 100 μM CHX for 2 hr, 81 nM PMA for 5 min or 3 μM IA23 for 5 min: rates of pronuclear formation were 54.4%, 84.3%, 34.2%, and 54.6%, respectively. More than 80% of pronucleate ova successfully developed into 2‐cell stage. Additive treatment with 5 mM SrCl2 for 1 hr had no positive effect on pronuclear formation. Incidences of aneuploidy (4.6%) and structural chromosome aberrations (1.0%) in parthenogenons produced by combined stimuli of IA23 and CHX were not significantly different from those (3.8% and 1.6%, respectively) in female pronuclei of ova fertilized in vitro, showing that combined treatments with IA23 and CHX cause neither nondisjunction at the second meiotic division nor structural aberrations in MII chromosomes. The present technique for parthenogenetic activation of Chinese hamster oocytes may be useful as an assessment system to detect aneugenic and clastogenic effects of mutagens on mammalian oocytes. Mol. Reprod. Dev. 47:72–78, 1997. © 1997 Wiley‐Liss, Inc.

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Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide

Cited by 92 publications

References 39 publications

Parthenogenetic Development and Ploidy following Various Chemical Activation Regiments of Bovine Oocytes

Parthenogenetic Development and Ploidy following Various Chemical Activation Regiments of Bovine Oocytes

The Effect of Activation Protocols on the Development of Cloned Goat Embryos

Parthenogenetic activation of Chinese hamster oocytes by chemical stimuli and its cytogenetic evaluation

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