Somatic cell nuclear transfer (SCNT) embryos can develop during the preimplantation period; however, most of these die after implantation period. A transcription factor, Cdx2, promotes differentiation of extraembryonic tissues and appears to be involved in the segregation of inner cell mass (ICM) and trophectoderm (TE) in preimplantation embryos. So far, we have demonstrated that the expression of Cdx2 in mouse SCNT embryos is delayed and its expression level is significantly lower than that in intracytoplasmic sperm injection (ICSI) embryos. Moreover, the ectopic expression of Oct-3/4 was observed in the TE tissues of SCNT blastocysts, but not in ICSI blastocysts. Fibroblast growth factor (FGF) receptor 2 (FGFR2) is specifically expressed in 8-cell to morula-stage embryos and trophectoderm (TE) and is essential for implantation; however, FGFR2 expression in SCNT embryos significantly decreases compared with IVF embryos. Therefore, it is likely that abnormality of differentiation that is controlled in development of pre-implantation in SCNT embryos leads to a rapid decrease of subsequent developmental ability. Then, we investigated the effects of FGF4 on development of SCNT embryos. Mouse SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998). B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. Data were analyzed by Student’s t-test. First, the timing to start adding FGF4 was decided by FGFR2 expression time about 54 h after cell injection and treated for 3, 6, 12, 24, and 42 h thereafter. In the case of FGF4 concentration at 25 ng mL-1 with treating time of 6 h from the 4- to 8-cell stages, SCNT embryos significantly promoted the development to morula and blastocyst stages (91 and 45%, respectively) compared with IVF embryos (80 and 30%, respectively; P < 0.05). However, longer treatment of 42 h with FGF4 made their morphology considerably worse. Then, concentrations of FGF4 at 5, 25, 50, 250, and 500 ng mL-1 with treating time of 6 h was examined. In case of FGF4 concentration at 25 and 50 ng mL-1, SCNT embryos significantly promoted the development to morula and blastocyst stages (P < 0.05). Immunohistochemical analysis showed segregation of the expression of Oct-3/4 and Cdx2 in ICM and TE, respectively, in FGF4-treated SCNT embryos, unlike in the case of nontreated SCNT embryos, which showed an ectopic expression of Oct-3/4 in TE tissues. Furthermore, after the transplantation of SCNT embryos treated with FGF4 at 50 ng mL-1 and the treating time of 6 h to recipient mice, most of the transferred embryos implanted and cloned mice were successfully produced as well as nontreated SCNT embryos. Therefore, FGF4 facilitates the development of SCNT embryos especially to the morula and blastocyst stages.
This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.
Effect of D-Glucuronic Acid and N-acetyl-D-Glucosamine Treatment during In Vitro Maturation on Embryonic Development after Parthenogenesis and Somatic Cell Nuclear Transfer in Pigs