Analysis of Oct-4 Expression and Ploidy in Individual Human Blastomeres

Abstract

Objective: Oct-4, a marker for totipotent cells, is upregulated by 10+ fold in the inner cell mass (ICM) of human blastocysts. Different levels of Oct-4 expression in the blastomeres of 4–8 cell embryos might predict development towards ICM (Oct-4 +) or trophectoderm (Oct-4 −). The object of this study is to determine simultaneously Oct-4 expression and chromosome number in individual blastomeres of human embryos.Design: Human blastomeres were individually separated into nuclear and cytoplasmic lysate fractions. The nucleus was submitted to FISH for chromosomes X, Y and 18 and the lysate was analyzed by RT-PCR for Oct-4 expression.Materials and Methods: Fifteen embryos (4–8 cells) were discarded with patient consent on day 3 or 4 and dissociated individually into 85 blastomeres by removing the zona pellucida with Acid Tyrode’s solution. Individual blastomeres were incubated in a cell membrane lysis buffer; the nucleus was identified by phase contrast microscopy, transferred to a slide, fixed with methanol:acetic acid (3:1) and subjected to FISH using X, Y and 18 CEP direct-labeled probes (Vysis) for 30min in HYBrite. Oct-4 expression was assessed by submitting the cytosol to single cell, nested RT-PCR reaction (Gibco BRL; Boehringer Mannheim). The sensitivity of this assay was determined by a dilution series of positive blastomeres; β-actin served as a control gene.Results: β-Actin was expressed in 94% of the blastomeres tested and 46% of these were also positive for Oct-4. In these blastomeres Oct-4 expression was up-regulated by a mean of 10-fold. The frequency of cells expressing Oct-4 decreased with increasing blastomere number. Although all embryos derived from 2pn zygotes, only 60% of the blastomeres were euploid. Oct-4 expression was detected in euploid and in multinucleated blastomeres. Oct-4 was also detected in 2 of 3 cells with an excess chromosome X or 18 but not in 5 cells that lacked a normal complement of chromosomes X, Y or 18.Conclusions: Individual blastomeres can be evaluated for Oct-4 expression and ploidy thereby providing the means for a more complete understanding of mosaic aneuploidy. Significantly, the nuclear and cytosolic fractions can also be assayed for any combination of ploidy, genetic mutations and their respective expression vectors by simultaneous FISH, genomic PCR and RT-PCR, respectively. In addition, further characterization of gene expression in the cytosolic fractions of Oct-4 + and Oct-4 − cells will provide information about key steps in differentiation during early embryonic development. Objective: Oct-4, a marker for totipotent cells, is upregulated by 10+ fold in the inner cell mass (ICM) of human blastocysts. Different levels of Oct-4 expression in the blastomeres of 4–8 cell embryos might predict development towards ICM (Oct-4 +) or trophectoderm (Oct-4 −). The object of this study is to determine simultaneously Oct-4 expression and chromosome number in individual blastomeres of human embryos. Design: Human blastomeres were individually separated into nuclear and cytoplasmic lysate fractions. The nucleus was submitted to FISH for chromosomes X, Y and 18 and the lysate was analyzed by RT-PCR for Oct-4 expression. Materials and Methods: Fifteen embryos (4–8 cells) were discarded with patient consent on day 3 or 4 and dissociated individually into 85 blastomeres by removing the zona pellucida with Acid Tyrode’s solution. Individual blastomeres were incubated in a cell membrane lysis buffer; the nucleus was identified by phase contrast microscopy, transferred to a slide, fixed with methanol:acetic acid (3:1) and subjected to FISH using X, Y and 18 CEP direct-labeled probes (Vysis) for 30min in HYBrite. Oct-4 expression was assessed by submitting the cytosol to single cell, nested RT-PCR reaction (Gibco BRL; Boehringer Mannheim). The sensitivity of this assay was determined by a dilution series of positive blastomeres; β-actin served as a control gene. Results: β-Actin was expressed in 94% of the blastomeres tested and 46% of these were also positive for Oct-4. In these blastomeres Oct-4 expression was up-regulated by a mean of 10-fold. The frequency of cells expressing Oct-4 decreased with increasing blastomere number. Although all embryos derived from 2pn zygotes, only 60% of the blastomeres were euploid. Oct-4 expression was detected in euploid and in multinucleated blastomeres. Oct-4 was also detected in 2 of 3 cells with an excess chromosome X or 18 but not in 5 cells that lacked a normal complement of chromosomes X, Y or 18. Conclusions: Individual blastomeres can be evaluated for Oct-4 expression and ploidy thereby providing the means for a more complete understanding of mosaic aneuploidy. Significantly, the nuclear and cytosolic fractions can also be assayed for any combination of ploidy, genetic mutations and their respective expression vectors by simultaneous FISH, genomic PCR and RT-PCR, respectively. In addition, further characterization of gene expression in the cytosolic fractions of Oct-4 + and Oct-4 − cells will provide information about key steps in differentiation during early embryonic development.