Transcriptome analysis of egg viability in rainbow trout, Oncorhynchus mykiss

Abstract

Background: Maternal transcripts are accumulated in the oocyte during oogenesis to provide for protein synthesis from oocyte maturation through early embryonic development, when nuclear transcription is silenced. The maternal mRNAs have short poly(A) tails after undergoing post-transcriptional processing necessary for stabilizing them for storage. The transcripts undergo cytoplasmic polyadenylation when they are to be translated. Transcriptome analyses comparing total mRNA and elongated poly(A) mRNA content among eggs of different quality can provide insight into molecular mechanisms affecting egg developmental competence in rainbow trout. The present study used RNA-seq to compare transcriptomes of unfertilized eggs of rainbow trout females yielding different eyeing rates, following rRNA removal and poly(A) retention for construction of the libraries.

Results: The percentage of embryos to reach the 32-cell stage at 24 h post fertilization was significantly correlated to family eyeing rate, indicating that inviable embryos were developmentally compromised before zygotic genome activation. RNA sequencing identified 2 differentially expressed transcripts (DETs) from total mRNA sequencing comparing females with low-quality (< 5% eyeing), medium-quality (30-50% eyeing), and high-quality (> 80% eyeing) eggs. In contrast, RNA sequencing from poly(A) captured transcripts identified 945 DETs between low- and high-quality eggs, 1012 between low- and medium-quality eggs, and only 2 between medium- and high-quality eggs. The transcripts of mitochondrial genes were enriched with polyadenylated transcript sequencing and they were significantly reduced in low-quality eggs. Similarly, mitochondrial DNA was reduced in low-quality eggs compared with medium- and high-quality eggs. The functional gene analysis classified the 945 DETs between low- and high-quality eggs into 31 functional modules, many of which were related to ribosomal and mitochondrial functions. Other modules involved transcription, translation, cell division, apoptosis, and immune responses.

Conclusions: Our results indicate that differences in egg quality may be derived from differences in maternal nuclear transcript activation and cytoplasmic polyadenylation before ovulation, as opposed to accumulation and storage of maternal nuclear transcripts during oogenesis. Transcriptome comparisons suggest low-quality eggs suffered from impaired oxidative phosphorylation and translation. The DETs identified in this study provide insight into developmental competence in rainbow trout eggs.

Keywords: Egg quality; Mitochondria; Polyadenylation; Rainbow trout; mRNA.

Fig. 1

Eyeing rates of the 192 surveyed rainbow trout families in the selective breeding program

Fig. 2

Relative abundance of mitochondrial DNA. Real-time quantitative PCR measurement of mt-atp6 and mt-cyb genes normalized to 18S, in low-, medium-, and high-quality eggs. (mean ± SEM)

Fig. 3

Gene transcripts detected in low-, medium-, and high-quality eggs. a Libraries constructed by rRNA removal. b Libraries constructed by poly(A) retention. Genes expressed with average normalized reads greater than 1 were counted

Fig. 4

Heat map of 50 differentially expressed transcripts. The top 10 up-regulated and 40 down-regulated genes in low-quality eggs were selected based on false discovery rate (FDR) value. The red bar indicates females with low-quality eggs and the green bar indicates females with high-quality eggs. * Indicates the gene has not been officially named. The gene name abbreviations are listed in Additional file 1: Table S7

Fig. 5

The 10 most represented gene ontology terms in biological process. The analysis included the 945 differentially expressed transcripts comparing low- and high-quality eggs. The number of significantly enriched gene ontology terms is shown in parenthesis, (P < 0.05)