139 INJECTION OF SOMATIC CELL CYTOPLASM INTO OOCYTES BEFORE ICSI IMPAIRED FULL-TERM DEVELOPMENT AND INCREASED PLACENTA WEIGHT IN MICE

During the process of somatic cell nuclear transfer, cytoplasm is introduced into the enucleated oocytes, in addition to the genomic material, regardless of the electrofusion methods (Wilmut et al. 1997) or direct injection of somatic nucleus by the Honolulu method (Wakayama et al. 1998). Only 1 to 2% of cloned embryos, however, develop to term with many incidences of developmental anomalies. These cloning failures may be explained by incomplete reprogramming of the donor cell genome, although it is not yet clear whether cytoplasmic materials of the somatic cell also have an affect on development of the cloned embryo. In an attempt to answer this question, this study investigates the effects of somatic cytoplasm of different mouse strains and cytoplasm of fertilized embryos at different stage by injecting them into intact mouse oocytes before intracytoplasmic sperm injection (ICSI). Mature oocytes collected from B6D2F1 female after 14 to 16 h of hCG injection were injected with (1) B6D2F1 cumulus cell cytoplasm with different volumes (collected by 2 to 3 ¼m of injection pipette and piezo pulses), (2) cumulus cell cytoplasm from different mouse strains (B6D2F1, ICR, C57BL/6), (3) cytoplasm of 1- to 8-cell embryos. After subsequent culture for 1 h, B6D2F1 sperm were injected into those oocytes and examined for preimplantation developmental competence. The total number of cells, inner cell mass (ICM), and expression of Oct4 in expanded blastocysts were also examined. In order to examine the effects of somatic cytoplasm on full-term development, we transferred 2-cell embryos at 24 h or morula and blastocysts at 72 h after ICSI to the oviduct or uterus of surrogate mothers (ICR) on Day 1 or 3 of pseudopregnancy. The control group received a sham injection with PVP before ICSI. The results showed that an increase the volume of cytoplasm from 1-fold to 4-fold (equivalent with the volume of 1 cumulus cell) resulted in impairing full-term development (28 and 7%, respectively, vs. 56 to 63% in the control group, P < 0.01). There was no difference in the frequency of embryos developing to the blastocyts stage between B6D2F1 and ICR somatic cytoplasms at the same volume. However, C57BL/6 somatic cytoplasm induced the 2-cell block to B6D2F2 embryos. Fertilized embryo cytoplasm did not reduce the frequency of blastocyst stage and full-term development. Interestingly, we found that somatic cytoplasm increased the placenta weight of ICSI embryo (0.2002 ± 0.03, n = 32; vs. 0.1198 ± 0.02 in control group, n = 87; P < 0.01). We also obtained placenta with no fetus when the volume of somatic cytoplasm was the same size as cumulus cell. We found that an increase in the volume of somatic cytoplasm led to low expression of Oct4 in expanded blastocysts. These findings indicated that injection of somatic cytoplasm into oocytes before ICSI decreased the preimplantation development, clearly impaired full-term development, and caused placental overgrowth in fertilized embryos. This study suggested that somatic cell cytoplasmic material is one cause of the low rate of full-term development of cloned animals.