148 MICE BLASTOCYST RECONSTRUCTION BY AGGREGATION OF INNER CELL MASS WITH TROPHECTODERM

Abstract

In order to reduce fetal losses of bovine clones the replacement of trophectoderm (TE) by microinjection of immunosurgically isolated inner cell mass (ICM) was proposed. It was reported that those techniques could damage both ICM and TE. An alternative to decrease this problem would be the technique of cell aggregation by approximation. Currently it has been used only in pre-compaction embryos. The aims of this study were to evaluate the effectiveness to aggregate post-compaction embryos and to verify the feasibility of reconstructed blastocysts production by ICM and TE approximation. Mice (Swiss Webster, SW; and C57BL/6/EGFP, EGFP) were superovulated to obtain embryos 2.5 to 3.5 days post-coitus. In both control groups (CG; exp. 1 and 2), whole embryos from 8 cells to early morula stages and without zona pellucida were aggregated. In all experiments, bisection was performed with a blade controlled by micromanipulator to produce two fragments according to the requirements of each experiment (i.e. demi embryos in Exp. 1 and Exp. 2 or TE fragment and whole ICM in Exp. 3). The joined fragments were in vitro cultured in microwell (300 µm diameter and based on well of the well system) filled with 400 µL of KSOMaa, covered with mineral oil by 24 h (incubation 37°C, 5% of CO₂ and saturated humidity). Embryonic aggregation rate (AR) was evaluated by detection of a single and cohesive cell mass (CG from Exp. 1 and Exp. 2) or by the presence of a typical morphologically blastocyst (all the remaining groups). Only to validate AR evaluation, 25% of the aggregation attempts were performed with distinct phenotype embryos (EGFP and SW) and they could be tracked under epifluorescence. In Exp. 1, the aggregation by approximation was tested to evaluate embryonic stages: 2 demi-blastocysts (2DB; n = 28), demi-morula and demi-blastocyst (DMDB; n = 20) and control group (CG; n = 25). In Exp. 2, an adhesive agent (phytohemagglutinin; PHA) and fragment increasing were used in the groups: 2DB (n = 24), 4 demi-blastocysts (4DB, that is four half-blastocyst) and CG (n = 22). After aggregation validation (Exp. 1 and Exp. 2), blastocyst reconstruction by the approximation of ICM and TE fragments (with PHA presence) was tested on groups: 1 TE fragment and 1 ICM (TI; n = 48) and 2 TE fragments and 1 ICM (2T1I; n = 17). The rates were analyzed by chi-square and Fisher’s exact tests (significance of 5%). In experiments 1 (3.6; 15.0; and 60.0%, respectively to 2DB, DMDB, and CG) and 2 (8.3; 36.4; and 77.3%, respectively to 2DB, 4DB, and CG), the AR differed among groups with exception of 2DB and DMDB (exp. 1; P > 0.05). In Exp. 3, there was no difference on the AR between TI and 2T1I (27.1 and 29.4%, respectively). Despite the low adhesion potential of the trophectoderm cells, is feasible to produce chimeric embryos by aggregation. We infer that the use of an adhesive agent could increase AR maintaining the contact of the embryonic fragments. The proposed technique was practical and effective for blastocyst reconstruction. Additionally, the present model established in mice is about to be tested in cattle for further validation.

Financial support: FAPESP.