Preimplantation mouse embryo biopsy methods and amplification of X-and Y-chromosome specific genes in single blastomeres for sex determination

Abstract

The purpose of the present study was to compare the efficiency of two different embryo biopsy methods regarding the development in vitro and in vivo of biopsied embryos at three different cleavage stages in the mouse model and to assess the reliability of sex determination in mouse preimplantation embryos using the two-step polymerase chain reaction method. In a comparative study, ICR preimplantation mouse embryos at different three stages (four-, eight-cell and early morula stages) were randomly allocated into four groups: (1) a control group, (2) a solution control group, (3) a PZD-push biopsy group, and (4) a direct aspiration biopsy group. The rates of normal blastocyst formation and hatching blastocysts of the biopsy embryos (including PZD-push and direct aspiration) were not significantly different from those of the control and solution control embryos. However, in the direct aspiration group, the rates of completely hatched blastocysts (4-cell: 67.4%, 8-cell: 72.8%, morula: 71.2% VS control embryos at the 4-cell: 86.7%, 8-cell: 86.4%, and morula: 87.6% and solution control group at the 4-cell: 85.4%, 8-cell: 85.2%, and morula: 85.2%) and live-births at the 4-cell (11.5% VS 27.3%) and 8-cell stages (24.2% VS 41.2%) were significantly (p<0.01) lower than those observed in the control and solution control embryos, whereas embryos, whereas embryo biopsy at the 8-cell and morula stages with PZD-push techniue were not significantly different from the controls. No grossly anatomical abnormalities were found in body weights or in subsequent ability to reproduce a second generation of mice derived from both biopsied and non-biopsy control embryos. Embryos biopsied at the morula stage had, however, more difficulties than at the 8-cell stage. Therefore, embryo biopsy at the 8-cell stage using PZD-push technique is the most suitable stage for embryo biopsy. For sex determination, the Sfy and Zfy genes, known to be presented in the sex-determining region of the mouse Y chromosome, were selected for Y-specific target sequences and the DXNds 3 locus located on the mouse X chromosome served as the internal control sequence. DNAs extracted from heart blood of male and female mice were used to test the accuracy and specificity of the selected primers using the two-step PCR method. The same experimental conditions were then applied to amplify the single copy genes in single mouse blastomere with two pairs of primers for each of the target sequences. The sex-determined embryos were transferred to the uteri of pseudopregnant recipients to test the consistency of the assay system. All male and female blood DNA sample results confirmed the correct sex indentification of the origin (100%). Nineteen of 20 single blastomeres showed the accurate diagnosis when compared with their 7/8 embryos. The sex of 36 out of 37 mouse pups born from biopsied male and female embryos agreed with the predicted sex. Reliable genetic analysis of sex chromosome specific sequences in single cells is possible by two-step PCR and may be applied for sex determination of human embryos and the diagnosis of defective genes in human preimplantation embryos derived from the in vitro fertilization program.