191 SEARCHING FOR THE IN VIVO TRANSCRIPTOME BLUEPRINT OF COMPETENT BOVINE OOCYTES

Abstract

Gene expression in early stage embryos relies mostly on post-transcriptional control of maternal transcripts accumulated during oocyte maturation. However, while the building process to obtain a competent oocyte is now better understood, it is still not clear what transcriptome blueprint composes a competent oocyte. The aim of the study was to compare the mRNA expression pattern between oocytes collected from fertile heifers and repeat breeders by using RNAseq. Oocytes were collected by ovum pickup from 3 heifers that were 11–15 months of age and became pregnant at the following oestrus and from 4 adult cows with an age of 4 to 7 years, classified as repeat breeders after they failed to become pregnant for a minimum of 3 consecutive AI. To obtain oocytes from follicles with the same degree of development, at time 0 all follicles visible through transrectal ultrasound examination were removed by transvaginal aspiration. Five days later oocytes were collected by ovum pick up from the newly formed follicles with diameters >5 mm. Oocytes from each animal were pooled and sequenced as a single sample. Total RNA was extracted by RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Amplified cDNA, was prepared starting from total RNA using the Ovation RNA-Seq System V2 (Nugen Technologies, San Carlos, CA, USA). After library preparation with TruSeq DNA Sample Prep kit (Illumina, Madison, WI, USA), sequencing was performed on an Illumina HiSEqn 2000. Galaxy and Chipster open web-based platforms were used to analyse the data. We identified 49 differentially expressed genes. Heifers’ oocytes mRNA pattern indicated greater potential to sustain cell division. In particular, oocytes expressed more Keratin 14 (a gene involved in cell proliferation) and kinesin family member 20B (a protein involved in cytokinesis). More competent oocytes also have a greater ability to repair single-strand breaks due to the high levels of endo/exonuclease (5′-3′), endonuclease G-like. This may reflect greater capacity to neutralise DNA damage and, therefore, greater ability to preserve and transmit high-quality DNA. Repeat breeders portray a different landscape; their greater expression of Jun oncogene, Heat shock protein 1, Stimulated by retinoic acid gene6, arylhydrocarbon receptor nuclear translocator, fibromodulin, and aryl-hydrocarbon receptor repressor suggest that these oocytes have been subjected to environmental stress during oocyte maturation. Their greater expressions of inhibin α, stearoyl-CoA desaturase, junctional adhesion molecule 2 have been previously shown to correlate with a reduced oocyte developmental potential. Furthermore, the Cannabinoid receptor protein 1 expression suggests a compromised ion function that can lead to a failed activation of the development program. Finally, the greater expression of Ubiquilin3 and Heat shock protein 1 led to high protein and mRNA degradation, respectively, suggesting that these oocytes are deprived of essential components to sustain embryo growth. In conclusion our data provide the first detailed snapshot of the mRNA pattern defining the differences between a competent oocyte and an incompetent oocyte in vivo.

Study supported by PRIN 2008, 2009 and EU-Quantomics.