Hostname: page-component-cd9895bd7-gvvz8 Total loading time: 0 Render date: 2024-12-18T17:45:56.228Z Has data issue: false hasContentIssue false
  • English
  • Français

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: [email protected].
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: [email protected].
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: [email protected].
Get access Rights & Permissions [Opens in a new window]

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

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

De Sousa, P.A., Dobrinsky, J.R., Zhu, J., Archibald, A.L., Ainslie, A., Bosma, W., Bowering, J., Bracken, J., Ferrier, P.M., Fletcher, J., Gasparrini, B., Harkness, L., Johnston, P., Ritchie, M., Ritchie, W.A., Travers, A., Albertini, D., Dinnyes, A., King, T.J. & 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, 642–50.CrossRefGoogle ScholarPubMed
Du, F., Shen, P.C., Xu, J., Sung, L.Y., Jeong, B.S., Lucky Nedambale, T., Riesen, J., Cindy Tian, X., Cheng, W.T., Lee, S.N. & Yang, X. (2006). The cell agglutination agent, phytohemagglutinin-L, improves the efficiency of somatic nuclear transfer cloning in cattle (Bos taurus). Theriogenology 65, 642–57.CrossRefGoogle ScholarPubMed
Enright, B.P., Kubota, C., Yang, X. & Tian, X.C. (2003). Epigenetic characteristics and development of embryos cloned from donor cells treated by trichostatin A or 5-aza-2′-deoxycytidine. Biol. Reprod. 69, 896–901.CrossRefGoogle ScholarPubMed
Enright, B.P., Sung, L.Y., Chang, C.C., Yang, X. & Tian, X.C. (2005). Methylation and acetylation characteristics of cloned bovine embryos from donor cells treated with 5-aza-2′-deoxycytidine. Biol. Reprod. 72, 944–8.CrossRefGoogle ScholarPubMed
Gibbons, J., Arat, S., Rzucidlo, J., Miyoshi, K., Waltenburg, R., Respess, D., Venable, A. & Stice, S. (2002). Enhanced survivability of cloned calves derived from roscovitine-treated adult somatic cells. Biol. Reprod. 66, 895–900.CrossRefGoogle ScholarPubMed
Gupta, M.K., Uhm, S.J., Han, D.W. & Lee, H.T. (2007a). Embryo quality and production efficiency of porcine parthenotes is improved by phytohemagglutinin. Mol. Reprod. Dev. 74, 435–44.CrossRefGoogle ScholarPubMed
Gupta, M.K., Uhm, S.J. & Lee, H.T. (2007b). Differential but beneficial effect of phytohemagglutinin on efficiency of in vitro porcine embryo production by somatic cell nuclear transfer or in vitro fertilization. Mol. Reprod. Dev. 74, 1557–67.CrossRefGoogle ScholarPubMed
Hashem, M.A., Bhandari, D.P., Kang, S.K. & Lee, B.C. (2007). Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica). Mol. Reprod. Dev. 74, 403–11.CrossRefGoogle ScholarPubMed
Hoshino, Y., Uchida, M., Shimatsu, Y., Miyake, M., Nagao, Y., Minami, N., Yamada, M. & Imai, H. (2005). Developmental competence of somatic cell nuclear transfer embryos reconstructed from oocytes matured in vitro with follicle shells inminiature pig. Cloning Stem Cells 7, 17–26.CrossRefGoogle ScholarPubMed
Im, G.S., Lai, L., Liu, Z., Hao, Y., Wax, D., Bonk, A. & Prather, R.S. (2004). In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres. Theriogenology 61, 1125–35.CrossRefGoogle ScholarPubMed
Kawahara, M., Wakai, T., Yamanaka, K., Kobayashi, J., Sugimura, S., Shimizu, T., Matsumoto, H., Kim, J.H., Sasada, H. & Sato, E. (2005). Caffeine promotes premature chromosome condensation formation and in vitro development in porcine reconstructed embryos via a high level of maturation promoting factor activity during nuclear transfer. Reproduction 130, 351–7.CrossRefGoogle Scholar
Kopecky, M., Semecky, V. & Nachtigal, P. (2005). Vimentin expression during altered spermatogenesis in rats. Acta Histochem. 107, 279–89.CrossRefGoogle ScholarPubMed
Li, G.P., Chen, D.Y., Lian, L., Han, Z.M., Zhu, Z.Y. & Seidel, G.E., Jr. (2002). Rabbit cloning: improved fusion rates using cytochalasin B in the fusion buffer. Mol. Reprod. Dev. 61, 187–91.CrossRefGoogle ScholarPubMed
Maguire, S.M., Tribley, W.A. & Griswold, M.D. (1997). Follicle-stimulating hormone (FSH) regulates the expression of FSH receptor messenger ribonucleic acid in cultured Sertoli cells and in hypophysectomized rat testis. Biol. Reprod. 56, 1106–11.CrossRefGoogle ScholarPubMed
Miyoshi, K., Yuki, Y. & Yoshida, M. (2005). Optimization of Ca2+ concentrations in fusion and activation media for production of cloned embryos from miniature pig somatic cells. J. Reprod. Dev. 51, 699–706.CrossRefGoogle ScholarPubMed
Miyoshi, K., Sato, K. & Yoshida, M. (2006). In vitro development of cloned embryos derived fromminiature pig somatic cells after activation by ultrasound stimulation. Cloning Stem Cells 8, 159–65.CrossRefGoogle ScholarPubMed
Ogura, A., Inoue, K. & Ogonuki, N. (2000). [Microinsemination and nuclear transfer using male germ cells]. Tanpakushitsu Kakusan Koso 45, 2021–9.Google ScholarPubMed
Romeo, R., Castorina, S. & Marcello, M.F. (1995). Intermediate filaments of human Sertoli cells in germinal alterations. Ital. J. Anat. Embryol. 100, 75–81.Google ScholarPubMed
Sung, L.Y., Shen, P.C., Jeong, B.S., Xu, J., Chang, C.C., Cheng, W.T., Wu, J.S., Lee, S.N., Broek, D., Faber, D., Tian, X.C., Yang, X. & Du, F. (2007). Premature chromosome condensation is not essential for nuclear reprogramming in bovine somatic cell nuclear transfer. Biol. Reprod. 76, 232–40.CrossRefGoogle Scholar
Vodicka, P., SmetanaK., Jr. K., Jr., Dvorankova, B., Emerick, T., Xu, Y.Z., Ourednik, J., Ourednik, V. & Motlik, J. (2005). The miniature pig as an animal model in biomedical research. Ann. N Y Acad. Sci. 1049, 161–71.CrossRefGoogle ScholarPubMed
Wakayama, T., Perry, A.C., Zuccotti, M., Johnson, K.R. & Yanagimachi, R. (1998). Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–74.CrossRefGoogle ScholarPubMed
Yamanaka, K., Sugimura, S., Wakai, T., Shoji, T., Kobayashi, J., Sasada, H. & Sato, E. (2007). Effect of activation treatments on actin filament distribution and in vitro development ofminiature pig somatic cell nuclear transfer embryos. J. Reprod. Dev. 53, 791–800.CrossRefGoogle Scholar
Yang, J., Yang, S., Beaujean, N., Niu, Y., He, X., Xie, Y., Tang, X., Wang, L., Zhou, Q. & Ji, W. (2007). Epigenetic marks in cloned rhesus monkey embryos: comparison with counterparts produced in vitro. Biol. Reprod. 76, 36–42.CrossRefGoogle Scholar