Effect of ICSI on gene expression and development of mouse preimplantation embryos

Abstract

Background: In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts.

Methods: We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSI(WM)) or KSOM medium with amino acids (ICSI(KSOMaa)) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip.

Results: Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOM(aa)).

Conclusions: Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.

Figure 1

Real-time PCR verification of gene expression data. Expression profile of brain derived neurotrophic factor (Bdnf), cyclin G1 (Ccng1), ubiquitin-conjugating enzyme E2A (Ube2a) and the nuclear receptor subfamily 3, group C, member 1 (Nr3c1) genes in murine blastocysts produced by in vivo fertilization and culture (in vivo), ICSI and IVC in KSOM with amino acids (ICSI_KSOMaa) and ICSI and IVC in WM (ICSI_WM) using real-time PCR technique (A) and microarray technique (B). Bars represent SD. Asterisk (*) indicates significant difference compare with in vivo at P < 0.05. Of note the genes tested by RT–PCR reflect the changes detected by microarray. The only difference is represented by the statistically significant (P < 0.05) increase after RT–PCR in Nr3c1 expression in ICSI_WM blastocysts, while the increase of Nr3c1 following microarray analysis in both media or after RT–PCR in ICSI_KSOMaa is not statistically significant.

Figure 2

Summary of up-regulated and down-regulated genes for the following comparisons: ICSI_KSOMaa versus in vivo, ICSI_WM versus in vivo, ICSI_KSOMaa versus ICSI_WM. Each comparison in the graph shows the number of genes statistically different when comparing the four replicates of one group with the four replicates of the other group. The numbers below each comparison indicate how many genes are statistically up-regulated or down-regulated (P < 0.05) more than 2-fold or 5-fold.

Figure 3

Hierarchical clustering with heat-map of ICSI_KSOMaa, ICSI_WM and in vivo embryos based on their gene expression profile. Unsupervised clustering was performed to analyze similarities among replicate samples across all treatment groups tested. Colors correspond to relative RNA abundance for the transcripts detected; each is represented by a horizontal bar in the heat-map. Yellow indicates high expression and blue denotes low expression.

Figure 4

A total number of differentially expressed genes in cellular, developmental and metabolic functional catergories overrepresented in ICSI_KSOMaa, ICSI_WM, and in vivo embryos (P < 0.05). Detail information of cellular, developmental and metabolic functions with differentially regulated genes in each comparison is listed in Supplementary data, Table S2.