Latrunculin A Dramatically Improves the Developmental Capacity of Nuclear Transfer Embryos Derived from Gene-Modified Clawn Miniature Pig Cells.

Abstract

Cells, tissues, and organs of gene-modified miniature pigs produced by somatic cell nuclear transfer (SCNT) are useful for xenotransplantation and biomedical research. However, the efficiency of SCNT still remains low, when the gene-modified cells are used as donor nuclei. The objective of this study is to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerisation inhibitor, on in vitro and in vivo developmental ability of cloned embryos derived from gene-modified miniature pig cells. Fetal fibroblasts were obtained from a female cloned fetus (Day 21 of pregnancy) of Clawn miniature pigs derived from porcine alpha 1-3 galactosyltransferase gene knockout cells. Pig oocytes with compact cumulus mass collected from ovaries of a local slaughterhouse were matured for 40-42 h and in vitro-matured oocytes were enucleated by aspiration with a thin glass pipette. Then a single fibroblast cell was introduced into the perivitelline space of an enucleated oocyte and electrically fused using needle-type electrodes in fusion medium. Following fusion treatment, reconstructed embryos were electrically activated. After activation, reconstructed embryos were cultured for 2 h in modified PZM3 supplemented with or without a cytoskeletal inhibitor [0.5 µM LatA, 1 µM LatA, or 10.4 µM cytochalasin B (CB)]. Following postactivation treatment, cloned embryos were cultured in modified PZM3 for further development and assessed for cleavage and blastocyst formation at 2 and 7 days of culture, respectively. To determine the in vivo developmental ability of cloned embryos, some embryos exposed to 0.5 µM LatA were transferred to the oviducts of a recipient miniature pig (24 h of culture) and lapalotomy was carried out on Day 21 of pregnancy. The cleavage rate was significantly higher (P<0.05) in embryos exposed to 0.5 µM LatA (86.7%) than those in embryos exposed to CB (74.1%) and without a cytoskeletal inhibitor (70.9%). Moreover the blastocyst formation rate was significantly higher (P<0.05) in embryos exposed to 0.5 µM (33.7%) or 1 µM LatA (32.9%) than those in embryos exposed to CB (18.5%) and without a cytoskeletal inhibitor (11.4%). In addition, five fetuses were obtained from recipient uteri after embryo transfer. The results of this study show for the first time that postactivation treatment with LatA is effective to improve the in vitro developmental capacity of gene-modified cloned miniature pig embryos and the embryos treated with LatA have the ability to develop into fetuses. (poster)