Purpose:We used transcriptome sequencing to characterize CL function during diestrus in non-pregnant female dogs. The aim of this study was to identify genes (and their respective pathways) that are differentially expressed (DE) during diestrus in order to obtain a broader understanding of CL function and developmental control.
Methods:The CL were obtained from non-pregnant bitches (n=30) on days 10, 20, 30, 40, 50 and 60 (n=3/group) post-ovulation (p.o.). Sequencing was performed on an Illumina HiSeq 2000 using the paired-end reads protocol and the Illumina TruSeq PE cluster kit v3-cBotHS (Illumina). The reads were mapped against the reference genome (Canis_familiaris. CanFam 3.1.75.dna.toplevel.fa) using TopHat v2.0.9, and the transcripts were assembled using Cufflinks and identified by their Ensembl name. The relative abundance of transcripts of RNA-seq fragments was measured by Cufflinks in fragment per kilobase of exon model per million mapped reads (FPKM). We performed real-time PCR (qPCR) to validate the RNAseq results using Taqman assays.
Results: A total of 771,208,718 reads (approximately 42 million per sample) was generated, with size of 100 bp. The software TopHat v2.0.9 assigned the reads to 34,408 genes, of which 9,000 were not annotated in the canine genome. 5,116 genes were differentially expressed (DEGs), although 1,106 of those were not yet annotated in the canine genome. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that many of the DE genes were related to cell proliferation, cell survival, angiogenesis, and immune system, which may regulate luteal formation and regression.
Conclusion: This study provides fundamental data that could prove useful in reproductive research regarding the regulation of CL lifespan.
Overall design: Corpus luteum mRNA profiles collected during diestrus on days 10,20,30,40,50 and 60 post-ovulation (n=3/group) were generated by deep sequencing, using Illumina HiSeq 2000.
Less...