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Fibroblasts from the new-born male testicle of Guangxi Bama mini-pig (Sus scrofa) can support nuclear transferred embryo development in vitro

Published online by Cambridge University Press:  01 May 2009

Hong-Bo Liu ,
Pei-Ru Lv ,
Xiao-Gan Yang ,
Xiao-E Qin ,
Dao-Yuan Pi ,
Yang-Qing Lu ,
Ke-Huan Lu ,
Sheng-Sheng Lu  and
Dong-sheng Li
Hong-Bo Liu
Affiliation:
Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Faculty of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China. Hubei Key Laboratory of Embryonic Stem Cell Research, Tai-He Hospital, Yunyang Medical College, 32S. Renmin Road, Shiyan, Hubei 442000, China.
Pei-Ru Lv
Affiliation:
Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Faculty of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China. Hubei Key Laboratory of Embryonic Stem Cell Research, Tai-He Hospital, Yunyang Medical College, 32S. Renmin Road, Shiyan, Hubei 442000, China.
Xiao-Gan Yang
Affiliation:
Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Faculty of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China.
Xiao-E Qin
Affiliation:
Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Faculty of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China.
Dao-Yuan Pi
Affiliation:
Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Faculty of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China.
Yang-Qing Lu
Affiliation:
Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Faculty of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China.
Ke-Huan Lu*
Affiliation:
Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Faculty of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China.
Sheng-Sheng Lu*
Affiliation:
Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Faculty of Animal Science and Technology, Guangxi University, Nanning, Guangxi 530004, China.
Dong-sheng Li*
Affiliation:
Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Hubei Key Laboratory of Embryonic Stem Cell Research, Tai-He Hospital, Yunyang Medical College, 32S. Renmin Road, Shiyan, Hubei 442000, China.
*
All correspondence to Ke-Huan Lu, Sheng-Sheng Lu and Dong-sheng Li, Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Tel/Fax: +86 771 3238064. e-mail: khlu@gxu.edu.cn.
All correspondence to Ke-Huan Lu, Sheng-Sheng Lu and Dong-sheng Li, Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Tel/Fax: +86 771 3238064. e-mail: khlu@gxu.edu.cn.
All correspondence to Ke-Huan Lu, Sheng-Sheng Lu and Dong-sheng Li, Guangxi Key Laboratory of Subtropical Bio-Resource Conservation and Utilization, Guangxi University, Nanning, Guangxi 530004, China. Tel/Fax: +86 771 3238064. e-mail: khlu@gxu.edu.cn.
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Summary

Miniature pigs are valuable for research in xenotransplantation and as models for investigating human diseases. Although many mammalian species have been cloned, the success rates have been very low, especially in the pig. In the present study, an attempt was made to optimize somatic cell nuclear transfer (SCNT) protocols for use in the production of the Guangxi Bama mini-pig. Firstly, mini-pig fibroblast cells from a new-born Guangxi Bama piglet were isolated and cultured. Cell type was identified by fluorescence immunocytochemistry (ICC); the cells expressed cimentin, but not cytoceratin and follicular stimulation hormone receptor (FSHR). Secondly, the optimal cell cycle synchronization protocol for treating fibroblast cells from the newborn piglet's testicle was investigated by contact inhibition and serum starvation. When fibroblast cells were treated by contact inhibition, a higher fusion (66.0% vs. 58.3%, p > 0.05) and blastocyst production (20.8% vs. 15.1, p > 0.05) rates were obtained than with serum starvation. Thirdly, to examine the ability of old cells to be morphologically remodelled after activation, testicular fibroblasts (passage 10–14) were introduced into enucleated oocytes; enlarged nuclei were formed in most of the reconstructed embryos at 6 h and enlarged nuclei or distinct pseudopronuclei were formed in nearly all the reconstructed embryos at 12 h. The old donor cell could be morphologically remodelled correctly and was competent to support embryo development to the blastocyst in vitro. Fourthly, the in vitro development potential of the cloned embryos was investigated using two types of donor cell: ear fibroblasts and low or high passage testicular fibroblasts. The rate of fusion was highest using low passage testicle fibroblasts (84.5% vs. 69.8% and 80.0%, p < 0.05), as was development to the blastocyst stage (14.6% vs. 7.7% and 6.3%, p < 0.05). Finally, the effect of phytohaemagglutinin (PHA) on parthenogenetic and cloned embryo development was examined. The PHA had no significant effect on the parthenogenetic embryos, but cloned embryo development to the blastocyst stage was significantly increased by PHA (10μg/ml), (13.4% vs. 5.6% and 5.6%, p < 0.05).

Keywords

Cell cycle synchronizationIndirect immunofluorescenceMini-pigPhytohaemagglutininSomatic cell nuclear transfer
Type
Research Article
Information
Zygote , Volume 17 , Issue 2 , May 2009 , pp. 147 - 156
Copyright
Copyright © Cambridge University Press 2009

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