Effects of 6-dimethylaminopurine, cytochalasin B and cycloheximide on in vitro development of I pig cloned embryos

Nguyen Khanh Van; Quan Xuan Huu; Vu Thi Thu Huong; Pham Doan Lan

doi:10.15625/1811-4989/18/3/14449

Author affiliations

Authors

Nguyen Khanh Van National institute of animal science
Quan Xuan Huu National institute of animal science
Vu Thi Thu Huong National Institute of Animal Science
Pham Doan Lan National institute of animal science

DOI:

https://doi.org/10.15625/1811-4989/18/3/14449

Keywords:

activation, 6-dimethylaminopurine, cytohalasin B, cycloheximide, pig oocytes, “I” SCNT embryos

Abstract

The present study was conducted to evaluate the effects of 6-dimethylaminopurine (6-DMAP), cytochalasin B (CyB) and cycloheximide (CHX) on in vitro development of “I” pig somatic cell nuclear transfer embryos. Oocytes after SCNT were activated with 2 mM 6-DMAP; 7.5 µg/ml CyB and 10 µg/ml CHX. The results indicated that the rate of oocytes no lysis in group activated by CHX was significantly lower compared with those in group activated by 6-DAMP and CyB (90.91% and 88.01%, respectively, P < 0,05). The blastocyst formation rate did not differ between group actvated by 6-DAMP and CyB (24.93% vs 24.4%, P > 0.05). Meanwhile the blastocyst formation rate in group activated by CHX was 15.01%, significantly lower than that in group activated by 6-DAMP and CyB (P < 0.05). The average number of cells in the blastocyst in group activated by 6-DAMP and CyB were higher than that in group activated by CHX (47.78, 44.57 vs 39.42, respectively). The use of 6-DMAP and CyB in the activation of pig oocytes after SCNT was more effective than CHX. The results of this study show that for the first time in Vietnam, we have created “I” pig SCNT embryo without zona pellucida with the rate of blastocyst formation from 15.01% to 24.93%.

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References

Akagi S., Adachi N, Matsukawa K, Kubo M and Takahashi S (2003) Developmental potential of bovine nuclear transfer embryos and postnatal survival rate of cloned calves produced by two different timings of fusion and activation. Mol Reprod Dev 66, 264-272.

Betthauser J, Forsberg E, Augenstein M, Chilids L, Eilertsen K, Enos J, Forsythe T, Golueke P, Jurgella G, Koppang R, Lesmeister T, Mallon K, Mell G, Misica P, Pace M, Pfister-Genskow M, Strelchenko N, Voelker G, Watt S, Thompson S, Bishop M (2000) Production of cloned pigs from in vitro system. Nat Biotechnol 18: 1055-1059.

Boquest AC, Grupen CG, Harrison SJ, Mcllfatrick SM, Ashman RJ, d,Apice AJF, Nottle MB (2002) Prodcution of cloned pigs from cultured fetal fibroblast cells. Biol Reprod; 66: 1283-1287.

Cheng WM, Sun XL, An L, Zhu SE, Li XH, Li Y and Tian JH (2007) Effect of different parthenogenetic activation methods on the developmental competence of in vitro matured porcine oocytes. Anim Biotechnol 18: 131-141.

De Sousa PA, Dobrinsky JR, Zhu J, Archibald AL, Ainslie A, Bosma W, Bowering J, Bracken J, Ferrier PM, Fletcher J, Gasparrini B, Harkness L, Johnston P, Ritchie M, Ritchie WA, Travers A, Albertini D, Dinnyes A, King TL and Wilmut I (2002) Somatic cell nuclear transfer in the pig, control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy. Biol Reprod 66: 642-650.

Grupen CG, Mau JC, McIlfatrick SM, Maddocks S, Nottle MB (2002) Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes. Mol Reprod Dev 62: 387-396.

Hosseini SM, Moulavi F, Asgari V, Shirazi A, Abazari-Kia A H, Ghanaei HR, Nasr-Esfahani MH (2013) Simple, fast, and efficient method of manual oocyte enucleation using a pulled Pasteur pipette. In vitro Cell Dev Biol – Animal DOI 10.1007/s11626-013-9630-4

Inoue K, Noda S, Ogonuki N, Miki H, Inoue S, Katayama K, Mekada K, Miyoshi H and Ogura A (2007) Differential developmental ability of embryos cloned from tissue-specific stem cells. Stem Cells 25: 1279-1285.

Kim YS, Lee SL, Ock SA, Balasubramanian S, Choe SY, Rho GJ (2005) Development of cloned pig embryos by nuclear transfer following different activation treatments. Mol Reprod Dev 70: 308-313.

Lee SH, Kim DY, Nam DH, Hyun SH, Lee GS, Kim HS, Lee CK, Kang SK, Lee BC and Hwang WS (2004) Role of messenger RNA expression of platelet activating factor and its receptor in porcine in vitro fertilized and cloned embryo development. Biol Reprod 71: 919-925.

Mario AMD, Masami S, Masumi K, Koji I and Yoshiyuki T (2003) Effects of cycloheximide treatment on in vitro development of porcine parthenotes and somatic cell nuclear transfer embryos. Jpn J Vet Res 50(4): 147-155.

Moses RM, Kline D, Masui Y (1995) Maintenance of metaphase in colcemid-treated mouse eggs by distinct calcium and 6-dimethylaminopurine (6-DMAP) sensitive mechanisms. Dev Biol 167: 329-337.

Nanassy L, Lee K, Javor A and Machaty Z (2007) Changes in MPF and MAPK activities in porcine oocytes activated by different methods. Theriogenology 68: 146-152.

Nanassy L, Lee K, Javor A and Machaty Z (2008) Effects of activation methods and culture conditions on development of parthenogenetic porcine embryos. Anim Reprod Sci 104: 264-274.

Park SJ, Koo OJ, Kwon DK, Gomez MNL, Kang JT, Atikuzzaman M, Kim SJ, Jang G and Lee BC (2010) Short-term treatment with 6-DMAP and demecolcine improves developmental competence of electrically or Thi/DTT-activated porcine parthenogenetic embryos. Zygote 19:1-8.

Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A and Campbell KH (2000) Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407, 86-90.

Roy PK, Kim G, Fang X, Hassan BMS, De. Soysa M, Shin ST and Cho JK (2017) Optimization of post-activation system to improve the embryonic development in porcine parthenogenesis and somatic cell nuclear transfer.. J Emb Trans Vol 32, No 3: 95-104.

Wakayama T and Yanagimachi R (2001) Mouse cloning with nucleus with nucleus donor cells of different age and type. Mol Reprod Dev 58: 376-383.

Wang L, Jiang H and Li Z (2016) Effects of electrical pulse and 6-DMAP on cleavage of golden hamster oocytes-Morphological and physiological observations. Journal of Functional Morphology and Kinesiology 1: 240-248.

Yang XY, Zhao JG, Li HW, Li H, Liu HF, Huang SZ and Zeng YT (2005) Improving in vivo development of cloned bovine embryos with hybrid (Holstein-Chinese Yellow) recipient oocytes recovered by ovum pick up. Theriogenology 64: 1263-1272.