Sex determines the expression level of one third of the actively expressed genes in bovine blastocysts

Abstract

Although genetically identical for autosomal Chrs (Chr), male and female preimplantation embryos could display sex-specific transcriptional regulation. To illustrate sex-specific differences at the mRNA level, we compared gene-expression patterns between male and female blastocysts by DNA microarray comparison of nine groups of 60 bovine in vitro-produced blastocysts of each sex. Almost one-third of the transcripts detected showed sexual dimorphism (2,921 transcripts; false-discovery rate, P < 0.05), suggesting that in the absence of hormonal influences, the sex Chrs impose an extensive transcriptional regulation upon autosomal genes. Six genes were analyzed by qPCR in in vivo-derived embryos, which displayed similar sexual dimorphism. Ontology analysis suggested a higher global transcriptional level in females and a more active protein metabolism in males. A gene homolog to an X-linked gene involved in network interactions during spliceosome assembly was found in the Y-Chr. Most of the X-linked-expressed transcripts (88.5%) were up-regulated in females, but most of them (70%) exhibited fold-changes lower than 1.6, suggesting that X-Chr inactivation is partially achieved at the blastocyst stage. Almost half of the transcripts up-regulated in female embryos exhibiting more than 1.6-fold change were present in the X-Chr and eight of them were selected to determine a putative paternal imprinting by gene expression comparison with parthenogenetic embryos. Five (BEX, CAPN6, BEX2, SRPX2, and UBE2A) exhibited a higher expression in females than in parthenotes, suggesting that they are predominantly expressed by the paternal inherited X-Chr and that imprinting may increase the transcriptional skew caused by double X-Chr dosage.

Fig. 1.

Comparison of male and female bovine blastocysts. (A) Hierarchical clustering of the 382 differentially expressed transcripts (Bonferroni correction) between male and female bovine blastocysts, comparing three different bulls and the three pools of embryos derived from Y- and X-sorted semen from each bull. The color gradient determines normalized gene expression of all of the samples. The dendrogram on the left depicts the grouping of samples based on the similarity between them. Samples were clearly grouped according to sex (blue and red bars on Left). (B) Chr distribution for the total transcripts present (green bars) and up-regulated in male (blue bars) or female (red bars) embryos (FDR P < 0.05). Percentages for each Chr out of the total transcripts with a known Chr location (7,691, 1,287, and 1,065 transcripts for present and up-regulated in males and females, respectively). (C) The pie chart shows the percentage of expressed X-linked transcripts (n = 218) grouped according to the fold change (FDR P < 0.05 correction). From pink to red were up-regulated in females (n = 193), blue were up-regulated in males (n = 3), and green did not show differences (n = 22). The line chart shows the percentage of X-linked transcripts compared to the total up-regulated transcripts in females with a known location after FDR for four groups according to the fold-change.

Fig. 2.

Relative mRNA abundance. (A) Relative poly(A) mRNA abundance of six genes (four X-linked -CAPN6, FMR1NB, SAT1 and UBE2A-, one Y-linked –YZRSR2-, and one autosomal -DNMT3A-) for male and female in vivo–derived blastocysts. (B) Relative poly(A) mRNA abundance of eight putative paternally expressed imprinted X-linked genes and one gene located on both X- and Y-Chrs (Y2467) for male (black bars), female (white bars), and parthenogenetic (dashed bars) in vitro blastocysts. Different letters indicate significant differences between groups based on one-way ANOVA (P ≤ 0.05).

Fig. 3.

Fatigo-based comparative analysis of gene ontology for the family of genes exhibiting sex-related transcriptional differences based on (A) molecular function, (B) biological function, and (C) cellular component.