P43. Several factors affect the developmental potential of rat somatic cell nuclear-transferred oocytes in vitro

Abstract

Background: Since the first successful report of cloned mammals after nuclear transfer of somatic cells in sheep in 1997, cloned animals of various species have been produced. Generally, somatic cell nuclear transfer (SCNT) is performed with non-activated metaphase II (MII) oocytes as recipients, because activation of oocytes has adverse effects on the developmental potential of SCNT oocytes. Rat oocytes are spontaneously activated soon after ovulation, but Zhou et al. successfully cloned the first rat by SCNT using the proteasome inhibitor MG132 to inhibit spontaneous oocyte activation (2003. Science 302, 1179). To date, however, this is the only report of a cloned rat by SCNT, indicating that successful cloning of rat SCNT depends on other unknown factors as well. Here we examined several factors affecting the developmental potential of rat SCNT embryos aimed at establishing reliable procedures for successful rat SCNT. Methods and results: First, we examined the effect of MG132 on spontaneous activation of rat oocytes. The oocytes (from 3- to 5-week-old Sprague Dawley rats) maintained developmental potential after parthenogenetic activation for at least 4 h in 5 µM MG132-supplemented medium. Long exposure of SCNT oocytes to MG132 before activation affected the developmental potential due to spindle abnormalities. SCNT oocytes receiving fresh cumulus cells as nuclear donors with short-duration exposure to MG132 had a high cleavage rate, and several oocytes developed to the blastocyst stage, but the rate was very low, with or without treatment with the histone deacetylase inhibitor Trichostatin A (TSA), which improves the developmental potential of SCNT oocytes in mouse and bovine. When donor cells were cultured before SCNT, reconstituted oocytes developed to the 4-cell stage at a higher rate than non-cultured oocytes, even those of the same cell origin, and some oocytes developed to the blastocyst stage. To improve the developmental potential of rat SCNT oocytes, not only should spontaneous activation be avoided, but donor preparation, epigenetic modification, and in vitro culture conditions should also be examined.