69 RELATION OF SPATIAL GENE EXPRESSION PATTERNS IN BOVINE EMBRYOS RECONSTRUCTED WITH SOMATIC CELLS TO BLASTOCYST DEVELOPMENT

Abstract

Recently, enhanced development to full term was obtained with embryos reconstructed with bovine early G₁ cells rather than with G₀ cells (Kasinathan et al. 2001 Nat. Biotechnol. 19, 1176-1178; Urakawa et al. 2004 Theriogenology 62, 714-728). However, the reason why donor somatic cells at the early G₁ phase are better for embryo reconstruction is unclear. In this study, we investigated the relation of spatial gene expression patterns at the 4- to 8-cell stage to blastocyst development of embryos reconstructed with early G₁ cells. Bovine fibroblasts stably transfected with ²-act/luc⁺/IRES/EGFP were used for embryo reconstruction. M phase cells were prepared as described by Urakawa et al. (2004). Early G₁ cells were obtained from cultured M phase cells soon after the M phase cells divided. Quiescent cells (cultured in 0.4% serum for 7 days) were used as G₀ cells for a control. The cells were electrofused with enucleated bovine oocytes matured in vitro, and activated with a calcium ionophore and cycloheximide. The reconstructed embryos were cultured until 60 hours post fusion (hpf), and zonae pellucidae of 4- to 8-cell embryos were removed by pronase. To determine gene expression, the LUC⁺ activity (luminescence) in the embryo blastomeres was detected with an imaging photon counter (Hamamatsu Photonics, Hamamatsu City, Shikuoka Prefecture, Japan) for 10 min. The embryos were categorized as being positive, mosaic, or negative depending on whether all, some or no blastomeres were luminescent, respectively. The embryos were cultured in mSOF medium individually until 168 hpf to assess development to the blastocyst stage. Blastocyst development of reconstructed embryos without detection of luminescence was also examined. Experiments were repeated three times, and the data were analyzed with Fisher's PLSD test following ANOVA. At 60 hpf, 75% (74/99) of embryos reconstructed with early G₁ cells and 55% (46/83) of embryos with G₀ cells developed to 4- to 8-cell stage embryos. The difference is significant (P < 0.05). The percentages of positive, mosaic, and negative embryos with G₁ cells were 49, 35 and 16%, and blastocyst rates were 30, 11, and 0%, respectively. With G₀ cells, the percentages were 32, 56, and 12%, and the blastocyst rates were 15, 4, and 0%, respectively. More positive embryos were obtained with early G₁ cells than with G₀ cells (P < 0.05). Blastocyst rates of the positive embryos with early G₁ cells were the same as with G₀ cells (P > 0.05). Blastocyst development of positive embryos was higher than that of mosaic and negative embryos in early G₁ and G₀ groups (P < 0.05). Without detection of luminescence, the blastocyst rates from the reconstructed embryos were 43% (35/81) and 16% (20/125) with early G₁ and G₀ cells, respectively (P < 0.05). These results suggest that the higher developmental capacity of embryos reconstructed with early G₁ cells might be related to the appropriate spatial gene expression at the 4- to 8-cell stage.

A part of this study was supported by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.