Volume 6, Issue 5 (7-2008)                   IJRM 2008, 6(5): 181-0 | Back to browse issues page

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Darabi M R, Nasr-Esfahani M H, Baharvand H, Mardani M, Karimi-Jashni H. Fusion and development of 2-cell bovine embryos to tetraploid blastocyst with different voltages and durations. IJRM. 2008; 6 (5) :181-0
URL: http://journals.ssu.ac.ir/ijrmnew/article-1-129-en.html
1- Department of Anatomy, Arak University of Medical Sciences, Arak, Iran
2- Department of Clinical and Experimental Embryology, Reproductive Medicine Research Center, Royan Institute, Isfahan campus, ACECR Tehran, Iran , mh.nasr-esfahani@royaninstitute.org
3- Department of stem cell, Royan Institute, Tehran, Iran
4- Department of Anatomy, Isfahan University of Medical Sciences, Isfahan, Iran
5- Department of Anatomy, Jahrom University of Medical Sciences, Jahrom, Iran
Abstract:   (848 Views)
Background: The values of embryonic stem cell and cloning are evident. Production of clone from embryonic stem cells can be achieved by introduction of stem cell into a tetraploid blastocyst. Tetraploid blastocyst can be produced in vitro by electrofusion of 2-cell embryos.
Objective: The aim of this study was to assess the effect of different voltages and durations on fusion rate of bovine 2-cell embryos and their subsequent development in vitro.   
Material and Methods: The in vitro produced bovine 2-cell embryos were categorized into 3 groups: (1) fused group (FG); 2-cell embryos fused by exposure to different voltages (0.5, 0.75, 1, 1.25 and 1.5 kV/cm) and durations (20, 40, 60, 80 and 100 μs), (2) exposed control group (ECG);  2-cell embryos exposed to different voltages and durations but remained unfused and (3) unexposed control group (UCG); embryos cultured without exposure to any voltage. The embryos from each group were cultured and fusion, cleavage and developmental rates were compared in each group.
Results: The results show that increased voltage, increases the fusion rate up to 88% for 1.5 kV/cm; however, the rate of cleavage and blastocyst formation decreases significantly to 18% and 10% respectively (p<0.05). Increased duration does not significantly increase fusion rate, however, in high voltage, increased duration decreases cleavage rate and blastocyst formation rate. Blastocyst formation rate in UCG showed a better development (32%) compared to FG (20%) or ECG (22.5%) (p<0.05).
Conclusion: It can be concluded that for optimal fusion, cleavage and development, one pulse of 0.75 kV/cm for 60μs should be applied.
Full-Text [PDF 124 kb]   (140 Downloads) |   |   Full-Text (HTML)  (73 Views)  
Type of Study: Original Article |

1. Nguyen VT, Sayaka W, Satoshi K, Hiroshi O, Takafusa H. Injection of somatic cell cytoplasm into oocytes before intracytoplasmic sperm injection impairs full-term development and increases placental weight in mice. Biol Reprod 2006; 74: 865-873. [DOI:10.1095/biolreprod.105.047803]
2. Shinozawa T, Sugawara A, Matsumoto A, Han Y-J, Tomioka I, Inai K, et al. Development of rat tetraploid and chimeric embryos aggregated with diploid cells. Zygot 2006; 14: 287-297 [DOI:10.1017/S096719940600387X]
3. Lan LI, Wei SHEN, Lingjiang MIN, Yujian SUN, Jixian DENG, Quingjie PAN. Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene. Front Biol Chaina 2008; 3: 269-274. [DOI:10.1007/s11515-008-0052-8]
4. Jingjuan JI, Tonghang GUO, Xianhong TONG, Lihua LUO, Guixiang ZHOU, Yingyun FU, et al. Experimental cloning of embryos through human-rabbit interspecies nuclear transfer. Front Biol Chaina 2007; 2: 80-84. [DOI:10.1007/s11515-007-0014-6]
5. Polejaeva IA, Campbell KHS. New advances in somatic cell nuclear transfer: Applications in transgenesis. Theriogenology 2000; 53: 117-126. [DOI:10.1016/S0093-691X(99)00245-9]
6. Skrzyszowska M, Smorag Z, Slomski R, Katska-Ksiazkiewicz L, Kalak R. Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning. Biol Reprod 2006; 74: 1114-1120. [DOI:10.1095/biolreprod.104.039370]
7. James RM, Klerkx A, Keighren M, Flockhart JH, West JD. Restricted distribution of tetraploid cells in mouse tetraploid-diploid chimaeras. Developmental Biology 1995; 167: 213- 226. [DOI:10.1006/dbio.1995.1018]
8. James RM, West JD. A chimaeric animal model for confined placental mosaicism. Human Genetic 1994; 93: 603-604. [DOI:10.1007/BF00202833]
9. Curnow EC, Gunn LM, Trounson AQ. Electrofusion of two-cell bovine embryos for the production of tetraploid blastocysts in vitro. Molecular Reproduction Development 2000; 56: 372-377. https://doi.org/10.1002/1098-2795(200007)56:3<372::AID-MRD7>3.0.CO;2-W [DOI:10.1002/1098-2795(200007)56:33.0.CO;2-W]
10. Berg H. Fusion of blastomeres and blastoctsts of mouse embryos. Bioelectrochemistry Bioenergetic 1982; 9: 223-228. [DOI:10.1016/0302-4598(82)80178-5]
11. Iwasaki S, Kono T, Fukatsu H, Nakahara T. Production of bovine tetraploid embryos by electrofusion and their developmental capacity in vitro. Gamete Research 1989; 24: 261-267. [DOI:10.1002/mrd.1120240303]
12. Tanghe S, Soom A, Mehrzad VJ, Maes D, Duchateau L, Kruif A. Cumulus contributions during bovine fertilization in vitro. Theriogenology 2003; 60: 135-149. [DOI:10.1016/S0093-691X(02)01360-2]
13. Khurana NK, Nieman H. Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos. Theriogenology 2000; 54: 741-756. [DOI:10.1016/S0093-691X(00)00387-3]
14. Gandolfi F, Luciano MA, Modina S, Ponzini A, Pocar P, Armstrong DT, et al. The in vitro developmental competence of bovine oocytes can be related to the morphology of the ovary. Theriogenology 1997; 48:1153-1160. [DOI:10.1016/S0093-691X(97)00348-8]
15. Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by heparin. Biol Reprod 1988; 38: 1171-1180. [DOI:10.1095/biolreprod38.5.1171]
16. Neglia G, Gasparrini B, Brienza VC, Palo RD, Campanile G, Presicce GA, et al. Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration. Theriogenology 2003; 59: 1123-1130. [DOI:10.1016/S0093-691X(02)01170-6]
17. Carolan C, Lonergan P, Langendonckt AV, Mermillod P. Factors affecting bovine embryo development in synthetic oviductal fluid following oocyte maturation and fertilization in vitro. Theriogenology 1995; 43: 1115-1128. [DOI:10.1016/0093-691X(95)00075-J]
18. Hevitt DA, England GCW. Synthetic oviductal fluid and oviductal cell co-cultue for canine oocyte maturation in vitro. Animal Reproduction Science 1999;55: 63-75 [DOI:10.1016/S0378-4320(98)00162-6]
19. Steeves TE, Gardner DK. Temporal and differential effects of amino acids on bovine embryo development in culture. Biol Reprod 1999; 61: 731-740. [DOI:10.1095/biolreprod61.3.731]
20. Yoshioka K, Othman AM, Taniguchi T, Yamanaka H, Sekikawa K. Differential pattern of blastulation in bovine morulae cultured in synthetic oviductal fluid medium containing FCS or BSA. Theriogenology 1997; 48: 997-1006. [DOI:10.1016/S0093-691X(97)00326-9]
21. Galli C, Duchi R, Crotti G, Turini P, Pondera N, Colleoni I, et al. Bovine embryo technologies. Theriogenology 2003; 59: 599-616. [DOI:10.1016/S0093-691X(02)01243-8]
22. Alexander K, Elena P, Loulia Z, Larissa V, Detlev G. Development of parthenogenetic rat embryos. Biol Reprod 2003; 68: 829-836. [DOI:10.1095/biolreprod.102.006494]
23. Gordon I. Culturing the early embryo. In: Laboratory Production of Cattle Embryo. 3rd Ed. Wallingford, CABI publication; 2003; 246-247.
24. Tarkowski AK. An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 1966; 5: 394-400. [DOI:10.1159/000129914]
25. Yoshizawa M, Konno H, Zhu S, Kageyama S. Chromosomal diagnosis in each individual blastomere of 5-to 10-cell bovine embryos derived from in vitro fertilization. Theriogenology 1999; 51: 1239-1250. [DOI:10.1016/S0093-691X(99)00068-0]
26. Lechniak D. The incidence of polyploidy and mixoploidy in early bovine embryos derived from in vitro fertilization. Genetic Sel Evolution 1996; 28: 321-328. [DOI:10.1186/1297-9686-28-4-321]
27. Chernomerdik LV, Sowers AE. Physical and ultrastructural evidence that integrin of the spectrin network controls the macroscopic fusion produce morphology following the electrofusion of erythrocyte ghosts. Biophysical Journal 1991; 60: 1026-1037. [DOI:10.1016/S0006-3495(91)82140-3]
28. Zhelev DV, Dimitrov D, Doinov SP. Correlation between physical parameters in electro fusion and electroporation of protoplasts. Bioelectrochemistry and Bioenergeti 1988; 20: 155-167. [DOI:10.1016/S0302-4598(98)80013-5]
29. Tatham BG, Pushett DA, Giliam KJ, Dowsing AT, Mahavorasilpa TL, Trounson AO. Electrofusion of in vitro produced bovine embryonic cells for the production of isofusion contours for cells used in nuclear transfer. Journal of Reproduction and Fertility Supplementary 1995; 49: 549-553.
30. Prochazka R, Vodicka P, Zodova D, Rybar R, Motlik J. Development of in vivo derived diploid and tetraploid pig embryos in a modified medium NCSU 37. Theriogenology 2004; 62: 155-164 [DOI:10.1016/j.theriogenology.2003.08.017]
31. Iwasaki S, Campbell HS, Galli S, Akiama K. Production of live calves derived from embryonic stem like cells aggregated with tetraploid embryos. Biol Repropd 2000; 62: 470-475. [DOI:10.1095/biolreprod62.2.470]
32. Xiangyun Li, Wei Wei, Jun Yong, Qing Jia, Yuansong Yu, Keqian Di. The genetic heterozygozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation. Reproduction 2005; 130: 53-59 [DOI:10.1530/rep.1.00667]
33. Iwasaki Shizue, Yasuko Ito, Iwasaki Setsuo. In-vitro development of aggregates of bovine inner cell mass cells or bovine mammary cells and putative tetraploid embryos produced by electrofusion. Journal of Reproduction and Development 1999; 45: 65-71 [DOI:10.1262/jrd.45.65]
34. Suzuki H, Ogasavara I, Takahashi H, Imada Y, Toyokava K. Electrofusion of blastomeres of hamster 2-cell embryos and dynamic changes of the cytoskeletal distribution. Journal of Reproduction and Development 2001; 47: 227-235. [DOI:10.1262/jrd.47.227]

Send email to the article author

© 2021 All Rights Reserved | International Journal of Reproductive BioMedicine

Designed & Developed by : Yektaweb