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• W1976376543 abstract "Irradiation of in-vitro-matured bovine oocytes with x-rays of different durations was performed to develop an alternative to conventional mechanical enucleation methods in somatic cell nuclear transfer. No significant difference in embryo development to the blastocyst stage was detected between nonmechanical and mechanical methods, and cytologic analyses of karyotype and microtubule formation showed the potential availability of x-ray irradiation. Irradiation of in-vitro-matured bovine oocytes with x-rays of different durations was performed to develop an alternative to conventional mechanical enucleation methods in somatic cell nuclear transfer. No significant difference in embryo development to the blastocyst stage was detected between nonmechanical and mechanical methods, and cytologic analyses of karyotype and microtubule formation showed the potential availability of x-ray irradiation. Since the first live animal was produced by somatic cell nuclear transfer (SCNT) (1Wilmut I. Schnieke A.E. McWhir J. Kind A.J. Campbell K.H. Viable offspring derived from fetal and adult mammalian cells.Nature. 1997; 385: 810-813Crossref PubMed Scopus (3988) Google Scholar), continuous attempts have been made to apply SCNT in biomedicine and biotechnology. Enucleation of oocytes to prepare them as recipients of donor somatic cells is usually performed by suction or squeezing of DNA materials with a micromanipulator. However, such techniques cannot be used in oocytes of every species owing to the induction of irreversible, mechanical damages during enucleation. A more gentle technique will be necessary for successful SCNT in oocytes having vulnerable cytoplasm and/or membranes. In one successful instance, Vajta et al. (2Vajta G. Lewis I.M. Trounson A.O. Purup S. Maddox-Hyttel P. Schmidt M et al.Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro.Biol Reprod. 2003; 68: 571-578Crossref PubMed Scopus (125) Google Scholar) recently developed a handmade cloning method without micromanipulation, but further study is needed to improve the efficiency of various SCNT programs. It has been reported that x-ray irradiation with an extremely low dose efficiently inhibited the proliferation of avian primordial germ cells without decreasing cell viability (3Li H.C. Kagami H. Matsui K. Ono T. Restriction of proliferation of primordial germ cells by the irradiation of Japanese quail embryos with soft X-rays.Comp Biochem Physiol A Mol Integr Physiol. 2001; 130: 133-140Crossref PubMed Scopus (27) Google Scholar). Although x-ray irradiation causes general disturbance in the living organism, the response to x-ray irradiation is dose-dependent in oocytes (4Lyon M.F. Phillips R.J. Specific locus mutation rates after repeated small radiation doses to mouse oocytes.Mutat Res. 1975; 30: 375-382Crossref PubMed Scopus (4) Google Scholar). Consequently, we evaluated whether x-ray irradiation before SCNT could be substituted for mechanical enucleation of oocytes. The bovine system was selected as the model in this study. Bovine fetal fibroblasts retrieved from a day-40 fetus were used as somatic cell donors after being trypsinized, subcultured (6–8 times), and stored at −196°C before SCNT (5Cho J.K. Lee B.C. Park J.I. Lim J.M. Shin S.J. Kim K.Y et al.Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation.Theriogenology. 2002; 57: 1819-1828Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Cumulus-oocyte complexes cultured for 22 hours were used as the recipient cytoplasts. Either mechanical enucleation or x-ray irradiation without enucleation was conducted before SCNT. In the case of the mechanical method, the zona pellucida was partially dissected at the equatorial area between the first polar body and the opposing pole, and the polar body and its surrounding cytoplasm (presumably containing DNA materials) were removed through this slit by squeezing with a holding pipette. In x-ray irradiation, oocytes were freed from cumulus cells by 0.1% (vol/wt) hyaluronidase solution. Oocytes were then placed in a 35-mm cell culture dish containing 1.5 mL of modified synthetic oviduct fluid medium (mSOF) (6Kim N.H. Funahashi H. Prather R.S. Schatten G. Day B.N. Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation.Mol Reprod Dev. 1996; 43: 248-255Crossref PubMed Scopus (114) Google Scholar) supplemented with 25 mmol/L N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid and 0.8% (vol/wt) bovine serum albumin. Irradiation with 15 kV(p) x-ray for 0, 10, 20, 40, or 60 seconds was then conducted after oocytes were placed on the rotating specimen stage. After the enucleation or the irradiation, a frozen–thawed single fibroblast cultured for 1 or 2 days in Dulbecco's minimal essential medium supplemented with 0.5% (vol/vol) fetal bovine serum was then introduced into the perivitelline space of an oocyte. The couplets were fused with a double direct current pulse of 1.75 kV/cm for 15 microseconds in 0.26 mol/L mannitol solution with an electro cell manipulator (BTX 2001, San Diego, CA) with a 3.2-mm gap between the stainless steel wire electrodes. At 4 hours after fusion, reconstructed embryos were chemically activated with 5 μmol/L ionomycin for 4 minutes and subsequently cultured in mSOF medium containing 1.9 mmol/L dimethylaminopurine for 4 hours. Embryos were then cultured in mSOF supplemented with 0.8% BSA for an additional 162 hours at 39°C under 5% CO2 in air atmosphere. The development of reconstructed embryos to the 2-cell, 8-cell, 16-cell, morula, and blastocyst stages was monitored at 42, 66, 90, 114, and 162 hours of culture, respectively. Microtubules and DNA morphology up to 10 hours after fusion were observed by indirect immunocytochemical techniques (6Kim N.H. Funahashi H. Prather R.S. Schatten G. Day B.N. Microtubule and microfilament dynamics in porcine oocytes during meiotic maturation.Mol Reprod Dev. 1996; 43: 248-255Crossref PubMed Scopus (114) Google Scholar) with a confocal microscope (Carl Zeiss-LSM510; Biorad, Hercules, CA). Some blastocysts obtained in this study were subjected to karyotyping with an air-drying method (7Dyban A.P. An improved method for chromosome preparations from preimplantation mammalian embryos, oocytes or isolated blastomeres.Stain Technol. 1983; 58: 69-72Crossref PubMed Scopus (86) Google Scholar). A general linear model (PROC-GLM) in a commercial software program (SAS 8.1; SAS Institute, Cary, NC) was used for statistical analysis. When significant model (difference in embryo development among treatments) effect was detected by analysis of variance in the model, each treatment effect was compared by the least-squares method. A total of six attemptswith random distribution were conducted, and significant differences were defined as P<.05. A significant model effect of the treatments on preimplantation development was detected. However, no significant differences in embryo development to the 2-cell (68%–85%), 8-cell (58%–73%), and blastocyst (11%–28%) stages were detected between the mechanical and x-ray irradiations of 10, 20, 40, or 60 seconds (Table 1). Significant differences were observed in the comparison of mechanical enucleation with the 0-second irradiation (neither irradiation nor enucleation): decreased (P<.05) development to the 2-cell (50%), 4-cell (40%), and blastocyst (4%) stages.TABLE 1In vitro development of bovine clone embryos enucleated by x-ray irradiationaIrradiation with 15kV(p) x-ray was conducted. of different durations after somatic cell nuclear transfer of bovine fetal fibroblasts.Irradiation time (sec)No. of enucleated oocytesNo. (%)dPercentage of the number of oocytes being injected with donor cells. of clone embryos developedBeing injected with donor cellsReconstructed (%)cPercentage of the number of oocytes reconstructured successfully.2-cell4-cell8-cell16-cellMorulaBlastocyst[48][72][96][120][144][168]0bMechanical enucleation by squeezing of ooplasm was conducted as control treatment.5340 (75)34 (85)eValues with different superscript letters differ significantly (P<.05).29 (73)eValues with different superscript letters differ significantly (P<.05).23 (56)15 (38)15 (38)11 (28)eValues with different superscript letters differ significantly (P<.05).0 (sham control)5648 (86)24 (50)f19 (40)f16 (33)9 (19)7 (15)2 (4)f104533 (73)25 (76)eValues with different superscript letters differ significantly (P<.05).22 (67)eValues with different superscript letters differ significantly (P<.05).19 (58)12 (36)5 (15)4 (12)eValues with different superscript letters differ significantly (P<.05).204133 (80)25 (76)eValues with different superscript letters differ significantly (P<.05).19 (58)eValues with different superscript letters differ significantly (P<.05).16 (48)10 (21)6 (18)4 (12)eValues with different superscript letters differ significantly (P<.05).404937 (76)25 (68)eValues with different superscript letters differ significantly (P<.05).22 (60)eValues with different superscript letters differ significantly (P<.05).18 (49)15 (40)8 (22)4 (11)eValues with different superscript letters differ significantly (P<.05).604534 (76)23 (68)eValues with different superscript letters differ significantly (P<.05).22 (65)eValues with different superscript letters differ significantly (P<.05).18 (53)12 (35)8 (24)7 (21)eValues with different superscript letters differ significantly (P<.05).Note: Model effect of treatments in each parameter, which was indicated as P value, was .0117, .0285, .2135, .3947, .1348, and .0415, in the development to the 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages, respectively. Number in square brackets indicates time (hours) after reconstruction.a Irradiation with 15kV(p) x-ray was conducted.b Mechanical enucleation by squeezing of ooplasm was conducted as control treatment.c Percentage of the number of oocytes reconstructured successfully.d Percentage of the number of oocytes being injected with donor cells.e,f Values with different superscript letters differ significantly (P<.05). Open table in a new tab Note: Model effect of treatments in each parameter, which was indicated as P value, was .0117, .0285, .2135, .3947, .1348, and .0415, in the development to the 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages, respectively. Number in square brackets indicates time (hours) after reconstruction. A total of 10 blastocysts were karyotyped, and all had a normal number of bovine chromosomes. Immunohistochemical analysis showed that 83% of reconstructed embryos (10 of 12) formed normal chromosome plate and spindles of the stages between metaphase to telophase. Such morphology was not different from that in IVF embryos observed previously. The results of the present experiment show that a low dose of x-ray could be used as an alternative to mechanical enucleation in SCNT. Similar rates of blastocyst formation were observed between mechanical enucleation and x-ray irradiation of an optimal dose. Considerable numbers of embryos reconstructed after x-ray irradiation had a potential to normally develop: they formed a normal spindle during embryo development and had a normal karyotype. It has been reported that x-rays cause chromosome mutations in mammalian oocytes when irradiated onto guinea pigs and mice (4Lyon M.F. Phillips R.J. Specific locus mutation rates after repeated small radiation doses to mouse oocytes.Mutat Res. 1975; 30: 375-382Crossref PubMed Scopus (4) Google Scholar, 8Cox B.D. Lyon M.F. X-ray induced dominant lethal mutations in mature and immature oocytes of guinea-pigs and golden hamsters.Mutat Res. 1975; 28: 421-436Crossref PubMed Scopus (25) Google Scholar). However, the treatments conducted in the present study differed from those in previous studies: we exposed the x-ray onto the oocytes directly, not onto the whole body. Only recipient cytoplasm, not donor nucleus, affected the irradiation. The strength of x-ray irradiation was decreased to 15 kV(p), and a minimal exposure time was selected on the basis of preliminary results. The portable x-ray generator used in this study has a revolving stage for the specimen, which enabled us to irradiate a large number of recipient oocytes very quickly and accurately. Nevertheless, these factors could not guarantee the avoidance of DNA damage in cloned embryos, and even retaining the normal chromosome number in cloned embryos after treatment did not fully show the chromosome normality. Supplemental results from karyotyping and confocal microscope analysis showed that, to some extent, x-ray irradiation might exert its effect upon chromosomes in an all-or-none manner, because developmental potential was not affected by exposure duration. This hypothesis is somewhat different from previous findings that dose response against x-ray irradiation was found in reproductive cells (4Lyon M.F. Phillips R.J. Specific locus mutation rates after repeated small radiation doses to mouse oocytes.Mutat Res. 1975; 30: 375-382Crossref PubMed Scopus (4) Google Scholar, 8Cox B.D. Lyon M.F. X-ray induced dominant lethal mutations in mature and immature oocytes of guinea-pigs and golden hamsters.Mutat Res. 1975; 28: 421-436Crossref PubMed Scopus (25) Google Scholar, 9Jaquet P. Saint-Georges L.D. Buset J. Baatout S. Vankerkom J. Baugnet-Mahieu L. Cytogenetic effects of X-rays in the guinea pig female germ cells II. The maturing oocyte.Mutat Res. 1997; 391: 193-199Crossref PubMed Scopus (16) Google Scholar), and this inconsistency might result from different doses of x-ray irradiation among studies. Although the use of x-ray irradiation does reduce mechanical damage resulting from alternative methods of enucleation, it might have other undesirable side effects, such as reducing the developmental competence of reconstructed embryos. Reduced development (from 28% to 11%) within the level of statistical insignificance might be owing to DNA mutation after the x-ray enucleation. Such effects might be supported by the findings of Jacquet et al. (9Jaquet P. Saint-Georges L.D. Buset J. Baatout S. Vankerkom J. Baugnet-Mahieu L. Cytogenetic effects of X-rays in the guinea pig female germ cells II. The maturing oocyte.Mutat Res. 1997; 391: 193-199Crossref PubMed Scopus (16) Google Scholar), who noted rapid elimination of meiotically competent oocytes from guinea pig ovaries by 1 or 2 Gy of x-ray. Apparently, large-scale experiments will be necessary to further confirm the safety of the nonmechanical x-ray irradiation method. In conclusion, this study showed the potential for x-ray irradiation to become an alternative to mechanical enucleation for the preparation of recipient oocytes. An x-ray-mediated, nonmechanical enucleation method has the potential to help not only in the development of novel SCNT techniques available for therapeutic cloning and bioreactor production, but also in the production of model animal clones derived from oocytes with extremely vulnerable cytoplasm. The evaluation of whether the x-ray enucleation could be applied for various SCNT protocols with other donor cell types, such as adult fibroblasts, might expand its feasibility. Further studies monitoring embryo development, chromosome normality, and long-term cytoskeletal dynamics after x-ray irradiation SCNT are essential for fully confirming the feasibility of this technique." @default.
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• W1976376543 title "Development of a nonmechanical enucleation method using x-ray irradiation in somatic cell nuclear transfer" @default.
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