Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

143 DEVELOPMENTAL EFFICIENCY IMPROVEMENTS OF NUCLEAR TRANSFER EMBRYOS BY REPROGRAMMING SOMATIC CELLS USING TESTIS-DERIVED CELL EXTRACTS

H.-Y. Choi, C. Won, B.-W. Kim, Y.-J. Kang, G.-H. Kang, Y. Oh, E.-R. Lee, S. Roh and S.-G. Cho

Reproduction, Fertility and Development 19(1) 189 - 189
Published: 12 December 2006

Abstract

Somatic cell cloning has promise for medical treatment using embryonic stem cells derived from cloned embryos. However, porcine cloning by somatic cell nuclear transfer has been inefficient and, even after birth, cloned pigs are found to carry a variety of abnormalities. Moreover, recent molecular analyses of cloned embryos have revealed abnormal epigenetic modifications. Therefore, the prevention of epigenetic errors is expected to lead to the improvement of the success rate in animal cloning. Reports of recent studies indicate that the direct transformation of one differentiated somatic cell type into another is possible and would be advantageous for producing isogenic replacement cells. Therefore, in this study, we modulated the cell fate of somatic donor cells by introducing cell extracts derived from porcine testis. Several porcine somatic cells, including primary and stabilized porcine fibroblasts or epithelial kidney cells, were treated with streptolysin O (SLO; 230 ng mL-1), which reversibly permeablizes plasma membrane, and incubated for 30 min with testis cell-derived cell extracts (4 mg mL-1). To reseal plasma membranes, cells were placed in DMEM containing 30% FBS and 2 mM CaCl2 for 30 min. After resealing the cell membranes, we incubated the cells for 3 weeks and analyzed the expression of testis-specific genes such as protamine 1, protamine 2, SOX 9, mullerian inhibitory substance (MIS), preproacrosine (ACR), phosphoglycerate kinase 2 (PGK-2), protein C, and c-kit ligand. In the reprogrammed primary porcine fibroblasts or epithelial kidney cells, the porcine testis extracts were able to activate the expression of the porcine testis sertoli cell-specific genes. The male germ cell functions were sustained for more than 10 days after the reprogramming process. Then, in vitro-matured oocytes were enucleated and a single cell (either reprogrammed or intact) was injected directly into cytoplasm of the oocytes. The reconstructed embryos were activated electrically and cultured in vitro for 7 days. The rate of blastocyst formation was significantly higher (P < 0.05; chi-square test) in the reprogrammed nuclear donor cells (27/119; 22.7 ± 5.0%) than in the control (intact) cells (11/83; 13.3 ± 3.2%). Taken together, our results suggest that testis-derived cell extracts can be successfully used to reprogram fibroblasts to express male germ cell function, thus improving the developmental efficiency of the nuclear transfer embryos.

https://doi.org/10.1071/RDv19n1Ab143

© CSIRO 2006

Committee on Publication Ethics

Export Citation Get Permission

Share

Share on Facebook Share on Twitter Share on LinkedIn Share via Email